Alkyl, alkenyl and alkynyl Chrysamine G derivatives for the antemortem diagnosis of Alzheimer&#39;s disease and in vivo imaging and prevention of amyloid deposition

ABSTRACT

Amyloid binding compounds which are non-azo derivatives of Chrysamine G, pharmaceutical compositions containing, and methods using such compounds to identify Alzheimer&#39;s brain in vivo and to diagnose other pathological conditions characterized by amyloidosis, such as Down&#39;s Syndrome are described. Pharmaceutical compositions containing non-azo derivatives of Chrysamine G and methods using such compositions to prevent cell degeneration and amyloid-induced toxicity in amyloidosis associated conditions are also described. Methods using non-azo Chrysamine G derivatives to stain or detect amyloid deposits in biopsy or post-mortem tissue are also described. Methods using non-azo Chrysamine G derivatives to quantify amyloid deposits in homogenates of biopsy and post-mortem tissue are also described.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/640,704, now abandoned, and also a continuation-in-part ofInternational Patent application, PCT/US96/05918, both filed May 1,1996, which is a continuation-in-part of U.S. patent application Ser.No. 08/432,019, now abandoned, filed May 1, 1995, which is acontinuation-in-part of U.S. patent application Ser. No. 08/282,289, nowabandoned, filed Jul. 19, 1994.

The present invention was made utilizing funds from the NationalInstitute of Ageing, grant numbers AG-05443, AG-05133 and AG-08974.

BACKGROUND OF THE INVENTION

The present invention relates to the identification of compounds thatare suitable for imaging amyloid deposits in living patients. Morespecifically, the present invention relates to a method of imagingamyloid deposits in brain in vivo to allow antemortem diagnosis ofAlzheimer's Disease. The present invention also relates to therapeuticuses for such compounds.

Alzheimer's Disease (“AD”) is a neurodegenerative illness characterizedby memory loss and other cognitive deficits. McKhann et al., Neurology34: 939 (1984). It is the most common cause of dementia in the UnitedStates. AD can strike persons as young as 40-50 years of age. Yet,because the presence of the disease is difficult to determine withoutdangerous brain biopsy, the time of onset is unknown. The prevalence ofAD increases with age, with estimates of the affected populationreaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551(1989); Katzman, Neurology 43: 13 (1993).

By definition, AD is definitively diagnosed through examination of braintissue, usually at autopsy. Khachaturian, Arch. Neurol. 42: 1097 (1985);McKhann et al., Neurology 34: 939 (1984). Neuropathologically, thisdisease is characterized by the presence of neuritic plaques (NP),neurofibrillary tangles (NFT), and neuronal loss, along with a varietyof other findings. Mann, Mech. Ageing Dev. 31: 213 (1985). Post-mortemslices of brain tissue of victims of Alzheimer's disease exhibit thepresence of amyloid in the form of proteinaceous extracellular cores ofthe neuritic plaques that are characteristic of AD.

The amyloid cores of these neuritic plaques are composed of a proteincalled the β-amyloid (Aβ) that is arranged in a predominatelybeta-pleated sheet configuration. Mori et al., Journal of BiologicalChemistry 267: 17082 (1992); Kirschner et al., PNAS 83: 503 (1986).Neuritic plaques are an early and invariant aspect of the disease. Mannet al., J. Neurol. Sci. 89: 169; Mann, Mech. Ageing Dev. 31: 213 (1985);Terry et al., J. Neuropathol. Exp. Neurol 46: 262 (1987).

The initial deposition of Aβ probably occurs long before clinicalsymptoms are noticeable. The currently recommended “minimum microscopiccriteria” for the diagnosis of AD is based on the number of neuriticplaques found in brain. Khachaturian, Arch. Neurol., supra (1985).Unfortunately, assessment of neuritic plaque counts must be delayeduntil after death.

Amyloid-containing neuritic plaques are a prominent feature of selectiveareas of the brain in AD as well as Downs Syndrome and in personshomozygous for the apolipoprotein E4 allele who are very likely todevelop AD. Corder et al., Science 261: 921 (1993); Divry, P., J.Neurol. Psych. 27: 643-657 (1927); Wisniewski et al., in Zimmerman, H.M. (ed.): PROGRESS IN NEUROPATHOLOGY, (Grune and Stratton, N.Y. 1973)pp. 1-26. Brain amyloid is readily demonstrated by staining brainsections with thioflavin S or Congo red. Puchtler et al., J. Histochem.Cytochem. 10: 35 (1962). Congo red stained amyloid is characterized by adichroic appearance, exhibiting a yellow-green polarization color. Thedichroic binding is the result of the beta-pleated sheet structure ofthe amyloid proteins. Glenner, G. N. Eng. J. Med. 302: 1283 (1980). Adetailed discussion of the biochemistry and histochemistry of amyloidcan be found in Glenner, N. Eng. J. Med., 302: 1333 (1980).

Thus far, diagnosis of AD has been achieved mostly through clinicalcriteria evaluation, brain biopsies and post mortem tissue studies.Research efforts to develop methods for diagnosing Alzheimer's diseasein vivo include (1) genetic testing, (2) immunoassay methods and (3)imaging techniques.

Evidence that abnormalities in Aβ metabolism are necessary andsufficient for the development of AD is based on the discovery of pointmutations in the Aβ precursor protein in several rare families with anautosomal dominant form of AD. Hardy, Nature Genetics 1: 233 (1992);Hardy et al., Science 256: 184 (1992). These mutations occur near the N-and C-terminal cleavage points necessary for the generation of Aβ fromits precursor protein. St. George-Hyslop et al., Science 235: 885(1987); Kang et al., Nature 325: 733 (1987); Potter WO 92/17152. Geneticanalysis of a large number of AD families has demonstrated, however,that AD is genetically heterogeneous. St. George-Hyslop et al., Nature347: 194 (1990). Linkage to chromosome 21 markers is shown in only somefamilies with early-onset AD and in no families with late-onset AD. Morerecently a gene on chromosome 14 whose product is predicted to containmultiple transmembrane domains and resembles an integral membraneprotein has been identified by Sherrington et al., Nature 375: 754-760(1995). This gene may account for up to 70% of early-onset autosomaldominant AD. Preliminary data suggests that this chromosome 14 mutationcauses an increase in the production of Aβ. Scheuner et al., Soc.Neurosci. Abstr. 21: 1500 (1995). A mutation on a very similar gene hasbeen identified on chromosome 1 in Volga German kindreds withearly-onset AD. Levy-Lahad et al., Science 269: 973-977 (1995).

Screening for apolipoprotein E genotype has been suggested as an aid inthe diagnosis of AD. Scott, Nature 366: 502 (1993); Roses, Ann. Neurol.38: 6-14 (1995). Difficulties arise with this technology, however,because the apolipoprotein E4 allele is only a risk factor for AD, not adisease marker. It is absent in many AD patients and present in manynon-demented elderly people. Bird, Ann. Neurol. 38: 2-4 (1995).

Immunoassay methods have been developed for detecting the presence ofneurochemical markers in AD patients and to detect an AD related amyloidprotein in cerebral spinal fluid. Warner, Anal. Chem. 59: 1203A (1987);World Patent No. 92/17152 by Potter; Glenner et al., U.S. Pat. No.4,666,829. These methods for diagnosing AD have not been proven todetect AD in all patients, particularly at early stages of the diseaseand are relatively invasive, requiring a spinal tap. Also, attempts havebeen made to develop monoclonal antibodies as probes for imaging of Aβ.Majocha et al., J. Nucl. Med., 33: 2184 (1992); Majocha et al., WO89/06242 and Majocha et al., U.S. Pat. No. 5,231,000. The majordisadvantage of antibody probes is the difficulty in getting these largemolecules across the blood-brain barrier. Using antibodies for in vivodiagnosis of AD would require marked abnormalities in the blood-brainbarrier in order to gain access into the brain. There is no convincingfunctional evidence that abnormalities in the blood-brain barrierreliably exist in AD. Kalaria, Cerebrovascular & Brain MetabolismReviews 4: 226 (1992).

Aβ antibodies are also disadvantageous for use in AD diagnostics becausethey typically stain deposits of Aβ containing non-β-sheet(non-fibrillar) Aβ in addition to the neuritic plaques. Yamaguchi etal., Acta Neuropathol., 77: 314 (1989). These deposits may be a separatetype of lesion, not necessarily involved in the dementing process of AD.The latter is suggested by findings of nonfibrillar amyloid deposits incognitively normal controls and aged dogs. Moran et al., MedicinaClinica 98: 19 (1992); Shimada et al., Journal of Veterinary MedicalScience 54: 137 (1992); Ishihara et al., Brain Res. 548: 196 (1991);Giaccone et al., Neurosci. Lett. 114: 178 (1990). Even if non-fibrillaramyloid deposits are forerunners of neuritic plaques, the keypathological event in AD may be the process that turns the apparentlybenign non-fibrillar amyloid deposit into the neuritic plaque with itsassociated halo of degeneration. Therefore, a probe is needed that isspecific for the fibrillar Aβ deposits and NFTs as a more specificmarker for AD pathophysiology than antibodies that would also labelnon-fibrillar amyloid deposits.

Recently, radiolabeled Aβ peptide has been used to label diffuse,compact and neuritic type plaques in sections of AD brain. Maggio etal., WO 93/04194. However, these peptides share all of the disadvantagesof antibodies. Specifically, peptides do not normally cross theblood-brain barrier in amounts necessary for imaging.

Congo red may be used for diagnosing amyloidosis in vivo in non-brainparenchymal tissues. However, Congo red is probably not suitable for invivo diagnosis of Aβ deposits in brain because only 0.03% of an injecteddose of iodinated Congo red can enter the brain parenchyma. Tubis etal., J. Amer. Pharm. Assn. 49: 422 (1960). Radioiodinatedbisdiazobenzidine compounds related to Congo red, such as Benzo Orange Rand Direct Blue 4, have been proposed to be useful in vitro and in vivoto detect the presence and location of amyloid deposits in an organ of apatient. Quay et al., U.S. Pat. Nos. 5,039,511 and 4,933,156. However,like Congo red, all of the compounds proposed by Quay contain stronglyacidic sulfonic acid groups which severely limit entry of thesecompounds into the brain making it extremely difficult to attain animaging effective quantity or detectable quantity in the brainparenchyma.

The inability to assess amyloid deposition in AD until after deathimpedes the study of this devastating illness. A method of quantifyingamyloid deposition before death is needed both as a diagnostic tool inmild or clinically confusing cases as well as in monitoring theeffectiveness of therapies targeted at preventing Aβ deposition.Therefore, it remains of utmost importance to develop a safe andspecific method for diagnosing AD before death by imaging amyloid inbrain parenchyma in vivo. Even though various attempts have been made todiagnose AD in vivo, currently, there are no antemortem probes for brainamyloid. No method has utilized a high affinity probe for amyloid thathas low toxicity, can cross the blood-brain barrier, and binds moreeffectively to AD brain than to normal brain in order to identify ADamyloid deposits in brain before a patient's death. Thus, no in vivomethod for AD diagnosis has been demonstrated to meet these criteria.

Very recent data suggest that amyloid-binding compounds will havetherapeutic potential in AD and type 2 diabetes mellitus. As mentionedabove, there are two broad categories of plaques in AD brain, diffuseand neuritic (classical). Diffuse plaques do not appear to inducemorphological reactions such as the reactive astrocytes, dystrophicneurites, microglia cells, synapse loss, and full complement activationfound in neuritic plaques. Joachim et al., Am. J. Pathol. 135: 309(1989); Masliah et al., loc. cit. 137: 1293 (1990); Lue and Rogers,Dementia 3: 308 (1992). These morphological reactions all signify thatneurotoxic and cell degenerative processes are occurring in the areasadjacent to the fibrillar Aβ deposits of neuritic plaques. Aβ-inducedneurotoxicity and cell degeneration has been reported in a number ofcell types in vitro. Yankner et al., Science 250: 279 (1990); Roher etal., BBRC 174: 572 (1991); Frautschy et al., Proc. Natl. Acad. Sci. 88:83362 (1991); Shearman et al., loc. cit. 91: 1470 (1994). It has beenshown that aggregation of the Aβ peptide is necessary for in vitroneurotoxicity. Yankner, Neurobiol. Aging 13: 615 (1992). Differences inthe state of aggregation of Aβ in diffuse and neuritic plaques mayexplain the lack of neurotoxic response surrounding the diffuse plaque.Lorenzo and Yankner, Proc. Natl. Acad. Sci., 91: 12243 (1994). Recently,three laboratories have reported results which suggest that Congo redinhibits Aβ-induced neurotoxicity and cell degeneration in vitro.Burgevin et al., NeuroReport 5: 2429 (1994); Lorenzo and Yankner, Proc.Natl. Acad. Sci. 91: 12243 (1994); Pollack et al., Neuroscience Letters184: 113 (1995); Pollack et al., Neuroscience Letters 197: 211 (1995).The mechanism appears to involve both inhibition of fibril formation andprevention of the neurotoxic properties of formed fibrils. Lorenzo andYankner, Proc. Natl. Acad. Sci. 91: 12243 (1994). Congo red also hasbeen shown to protect pancreatic islet cells from the toxicity caused byamylin. Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994).Amylin is a fibrillar peptide similar to Aβ which accumulates in thepancreas in type 2 diabetes mellitus.

It is known in the art that certain azo dyes may be carcinogenic. Morganet al. Environmental Health Perspectives, 102(supp.) 2: 63-78, (1994).This potential carcinogenicity appears to be based largely on the factthat azo dyes are extensively metabolized to the free parent amine byintestinal bacteria. Cerniglia et al., Biochem. Biophys. Res. Com., 107:1224-1229, (1982). In the case of benzidine dyes (and many othersubstituted benzidines), it is the free amine which is the carcinogen.These facts have little implication for amyloid imaging studies in whichan extremely minute amount of the high specific activity radiolabelleddye would be directly injected into the blood stream. In this case, theamount administered would be negligible and the dye would by-pass theintestinal bacteria.

In the case of therapeutic usage, these facts have critical importance.Release of a known carcinogen from a therapeutic compound isunacceptable. A second problem with diazo dye metabolism is that much ofthe administered drug is metabolized by intestinal bacteria prior toabsorption. This lowered bioavailability remains a disadvantage even ifthe metabolites released are innocuous.

Thus, a need exists for amyloid binding compounds which are similar toCongo red but which enter the brain (Congo Red does not). Such compoundscould be used in preventing cell degeneration associated with fibrilformation and thereby treat pathological conditions in amyloidassociated diseases, such as AD and Downs Syndrome and in treatingpancreatic islet cell toxicity in type 2 diabetes mellitus.

A further need exists for amyloid binding compounds that are non-toxicand bioavailable and, consequently, can be used in therapeutics.

SUMMARY OF THE INVENTION

It therefore is an object of the present invention to provide a safe,specific method for diagnosing AD before death by in vivo imaging ofamyloid in brain parenchyma. It is another object of the presentinvention to provide an approach for identifying AD amyloid deposits inbrain before a patient's death, using a high-affinity probe for amyloidwhich has low toxicity, can cross the blood-brain barrier, and candistinguish AD brain from normal brain. It is another object to providea treatment for AD which will prevent the deposition or toxicity of Aβ.It is another object to provide a technique for staining and detectingamyloid deposits in biopsy or post-mortem tissue specimens. It isanother object to provide a method for quantifying amyloid deposition inhomogenates of biopsy or post-mortem tissue specimens.

In accomplishing these and other objects, there has been provided, inaccordance with one aspect of the present invention, an amyloid bindingcompound of Formula I or a water soluble, non-toxic salt thereof:

X is C(R″)₂

(wherein each R″ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O-CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂O(CO)R′, OR′, SR′, COOR′, R_(Ph), CR′═CR′—R_(Ph),CR′₂—CR′₂—R_(Ph) (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R″, a tri-alkyl tin, atetrazole or oxadiazole of the form:

 (wherein R′ is H or a lower alkyl group)

 or X is CR′═CR′, N═N, C═O, O, NR′ (where R′ represents H or a loweralkyl group), S, or SO₂;

each R₁ and R₂ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O) —R′, N(R′)₂, NO₂, (C═O)N(R′)₂O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin, R_(Ph), CR′═CR′—R_(Ph),CR′₂—CR′₂—R_(Ph) (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R, and R₂, a tetrazole oroxadiazole of the form:

(wherein R′ is H or a lower alkyl group), or a triazene of the form:

(wherein R₈ and R₉ are lower alkyl groups) or

each Q is independently selected from one of the following structures:

IA, IB, IC, ID, IE, IF and IG, wherein

IA has the following structure:

 wherein:

each of R₃, R₄, R₅, R₆, or R₇ independently is defined the same as R₁above;

IB has the following structure:

 wherein:

each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently is definedthe same as R₁ above;

IC has the following structure:

 wherein:

each of R₁₇, R,₁₈, R₁₉, R₂₀, or R₂₁ independently is defined the same asR₁ above;

ID has the following structure:

 wherein:

each of R₂₂, R₂₃, or R₂₄ independently is defined the same as R₁ aboveand

 represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

 wherein:

each of R₂₅, R₂₆, or R₂₇ independently is defined the same as R₁ aboveand

 represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

 wherein:

exactly one of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁ orR₃₂ independently is defined the same as R₁ above;

IG has the following structure:

 wherein:

exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈, or R₃₉ is the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈, or R₃₉ independently is defined the same as R₁ above.

It is another object of the present invention to provide an amyloidbinding compound of Formula I, as defined above, or a water soluble,non-toxic salt thereof, wherein at least one of the substituents R₁-R₇and R₁₀-R₃₉ is selected from the group consisting of ¹³¹I, ¹²³I, ⁷⁶Br,⁷⁵Br, ¹⁸F, ¹⁹F, ¹²⁵I, CH₂—CH₂—¹⁸F, O—CH₂—CH₂—¹⁸F, CH₂—CH₂—CH₂—¹⁸F,O—CH₂—CH₂-CH₂—¹⁸F and a carbon-containing substituent as specified inFormula I wherein at least one carbon is ¹¹C or ¹³C.

It is a further object of the invention to provide an amyloid bindingcompound of Formula I, as defined above, or a water-soluble, non-toxicsalt thereof, wherein the compound binds to Aβ with a dissociationconstant (K_(D)) between 0.0001 and 10.0 μM when measured by binding tosynthetic Aβ peptide or Alzheimer's Disease brain tissue.

Still another object of the present invention is to provide a method forsynthesizing an amyloid binding compound of Formula I, as defined above,or a water soluble, non-toxic salt thereof, wherein at least one of thesubstituents R₁-R₇ and R₁₀-R₃₉ is selected from the group consisting of¹³¹I, ¹²³I, ⁷⁶Br, 75Br, ¹⁸F and ¹⁹ ⁹F, comprising the step of reactingan amyloid binding compound of Formula I, as defined above, or a watersoluble, non-toxic salt thereof wherein at least one of the substituentsR₁-R₇ and R₁₀-R₃₉ is a tri-alkyl tin, with a halogenating agentcontaining ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹F.

An additional object of the present invention is a pharmaceuticalcomposition for in vivo imaging of amyloid deposits, comprising (a) anamyloid binding compound of Formula I, as defined above, or a watersoluble, non-toxic salt thereof, wherein at least one of thesubstituents R₁-R₇ and R₁₀-R₃₉ is selected from the group consisting of¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F and a carbon-containing substituent asspecified in Formula I wherein at least one carbon is ¹¹C or ¹³C, and(b) a pharmaceutically acceptable carrier.

Yet another object of the present invention is an in vivo method fordetecting amyloid deposits in a subject, comprising the steps of: (a)administering a detectable quantity of the above pharmaceuticalcomposition, and (b) detecting the binding of the compound to amyloiddeposit in said subject. It is also an object of the present inventionto provide an in vivo method for detecting amyloid deposits in a subjectwherein the amyloid deposit is located in the brain of a subject. Thismethod of the invention may be used in a subject who is suspected ofhaving an amyloidosis associated disease or syndrome selected from thegroup consisting of Alzheimer's Disease, Down's Syndrome, andhomozygotes for the apolipoprotein E4 allele.

Another object of the invention relates to pharmaceutical compositionsand methods of preventing cell degeneration and toxicity associated withfibril formation in amyloidosis associated conditions such as AD andType 2 diabetes mellitus. Such pharmaceutical compositions comprisealkyl, alkenyl and alkynyl derivatives of Chrysamine G and apharmaceutically acceptable carrier. Such compounds would be non-toxic.

Another object of the invention relates to the use of the probes as astain for the visualization and detection of amyloid deposits in biopsyor post-mortem tissue specimens.

Another object of this invention relates to the use of radiolabeledprobes for the quantitation of amyloid deposits in biopsy or postmortemtissue specimens.

Another object relates to a method of distinguishing Alzheimer's diseasebrain from normal brain.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the chemical structures of Chrysamine G and severalChrysamine G analogues or derivatives which have been synthesized andtested, including the 3-iodo derivative (3-ICG), the 3-iodo dimethoxyderivative (3-ICG(OMe)₂), the dimethyl ester derivative (CG(COOMe)₂),the phenol derivative, salicylic acid (SA), the aniline derivative(½CG), Congo red, the 3,3′-diiodo derivative (3,3′-I₂CG), the3,3′-dibromo derivative (3,3′-Br₂CG), the 3,31′-dichloro derivative(3,3′-Cl₂CG), the 3-bromo derivative (3-BrCG), and the 5-fluorosalicylicacid derivative ((5-FSA)CG). The numbers in the figure refer to eachcompound's K_(i) (in μM) for inhibition of [¹⁴C] Chrysamine G binding tothe synthetic peptide, Aβ(10-43).

FIG. 1B illustrates the chemical structures of several Chrysamine Ganalogues or derivatives which have been synthesized and tested,including the 3,3′-dicarboxylic acid derivative (3,3′-(COOH)₂CG), the2,2′-disulfonic acid derivative of Chrysamine G (2,2′-(SO₃)₂CG), the3-bromo, 3-isopropylsalicylic acid derivative (3-Br-(3-iPrSA)CG), the3-isopropylsalicylic acid derivative ((3-iPrSA)CG), the 2,4-diphenolderivative (2,4-Diphenol), the γ-resorcylic acid derivative((6-OHSA)CG), the 3,3′, 5,5′-tetramethylbenzidine derivative (3,3′,5,5′—(CH₃)₄CG), the 3,3′-dimethyl derivative (3,3′-(CH₃)₂CG), the2,2′-dimethyl derivative (2,2′-(CH₃)₂CG), the benzisoxazole derivative(CG Benzisoxazole), and the 3-carboxy alkyne derivative (3-(COOH)—C3C).The numbers in the figure refer to each compound's K_(i) (in μM) forinhibition of [¹⁴C] Chrysamine G binding to the synthetic peptide,Aβ(10-43).

FIGS. 2A-2K illustrate the chemical structures of the alkenyl derivativeof Chrysamine G and alkenyl tri-alkyl tin derivatives of analogues ofChrysamine G, in particular heterocyclic analogues. Note that thesestructures represent one-half of a molecule which is symmetric aroundthe wavy bond shown in the upper right, except that the tri-alkyl tinmoiety may only be on one side of the biphenyl group. The tri-alkyl tinderivatives are stable intermediate and immediate precursors for thepreparation of high specific activity halogenated radioactivederivatives. The heterocyclic analogues represent alternative means ofplacing weakly acidic moieties in the same structural position as themoderately acidic carboxylic acid group of Chrysamine G. These tri-alkyltin precursor compounds are shown in their protonated form, yet those ofskill in the art recognize that their deprotonated forms and tautomersalso are embraced by these drawings.

2A) alkenyl derivative of Chrysamine G.

2B) alkenyl tri-alkyl tin derivative of Chrysamine G;

2C) alkenyl tri-alkyl tin derivative of the 3-Hydroxy-1,2-benzisoxazoleanalogue;

2D) alkenyl tri-alkyl tin derivative of the phthalimide orisoindole-1,3(2H)-dione analogue;

2E) alkenyl tri-alkyl tin derivative of the phthalhydrazide or2,3-benzodiazine-1,4(2H,3H)-dione analogue;

2F) alkenyl tri-alkyl tin derivative of the2,3-benzoxazine-1,4(3H)-dione analogue;

2G) alkenyl tri-alkyl tin derivative of the(2H)1,3-benzoxazine-2,4(3H)-dione analogue; 2H) alkenyl tri-alkyl tinderivative of the (3H)2-benzazine-1,3(2H)-dione analogue;

2I) alkenyl tri-alkyl tin derivative of the 1,8-Naphthalimide analogue.

2J) alkenyl tri-alkyl tin derivative of the tetrazole analogue

2K) alkenyl tri-alkyl tin derivative of the oxadiazole analogue.

FIG. 3 Displacement curves of [¹⁴C] Chrysamine G binding to Aβ(10-43) byseveral structural analogues of Chrysamine G. Abbreviations refer tothose used in FIG. 1. FIG. 3A) Chrysamine G (open triangles); (5-FSA)CG(filled diamonds); 3,3′-(COOH)₂CG (filled squares); 2,2′-(SO₃)₂CG(filled circles). FIG. 3B) Chrysamine G (open triangles); Congo red(open circles); aniline derivative (open inverted triangles); phenolderivative (open squares); salicylic acid (X's). Curves which showincreased binding at higher concentrations do so because of theformation of micelles. Bedaux, F. et al., Pharm. Weekblad 98: 189(1963).

FIG. 4A is a Scatchard plot of Chrysamine G binding to Aβ(10-43). Thecurved line represents a nonlinear least-squares fit to a two,independent binding site model. The straight lines represent theindividual components.

FIG. 4B is a Scatchard analysis of [¹⁴C]CG binding to typical control(diamonds) and AD brain samples (squares). The dashed line has the sameslope as the AD line and is meant to aid in the comparison with thecontrol slope. This AD brain sample had 48 NP/×200 magnification, aK_(D) of 0.35 μM, and a B_(max) of 790 fmol/μg protein. The control hada K_(D) of 0.48 μM, and a B_(max) of 614 fmol/μg protein.

FIG. 5 is a graph illustrating the linearity of the binding assay withrespect to peptide concentration. Approximately 0.9 μg of Aβ(10-43) wasused in the typical assay.

FIG. 6A is a graph illustrating the time course of association ofChrysamine G and Aβ(10-43).

FIG. 6B is the graphic illustration of the determination of theassociation rate constant (k₁).

FIG. 6C is a graph of the time course of dissociation of Chrysamine Gfrom Aβ(10-43).

FIG. 7 is a graphic representation of a molecular model of theinteraction between Chrysamine G and Aβ.

FIG. 8A is a graph illustrating the correlation between the amount of[¹⁴C] Chrysamine G bound and the number of neuritic plaques (NP) in ADbrain samples.

FIG. 8B is a graph illustrating the correlation between the amount of[¹⁴C] Chrysamine G bound and the number of neurofibrillary tangles (NFT)in AD brain samples. In both FIGS. 8A and 8B, the x-axis represents theaverage NP or NFT count per field at ×200 magnification in Bielschowskystained sections of either the superior/middle frontal (n=10) orsuperior temporal cortex (n=10). The filled symbols and heavy linesindicate brains without amyloid angiopathy, the open symbols and dashedlines indicate brains with amyloid angiopathy. The y-axis representstotal, absolute [¹⁴C] Chrysamine G binding (fmol/μg protein) inhomogenates of brain samples adjacent to those used for staining.Approximately 75 μg protein and 150 nM [¹⁴C] Chrysamine G were used.

FIGS. 9A, 9B and 9C. The binding of Chrysamine G to various brain areasin samples of AD brain having more than 20 NPs/×200 magnification,referred to as “High Plaque AD Brains”, is shown in FIG. 9A. The bindingof Chrysamine G to brain areas in samples of AD brain having less than20 NPs/×200 magnification, referred to as “Low Plaque AD Brains”, isshown in FIG. 9B. The data points represent the ratio of [¹⁴C]Chrysamine G binding in the designated brain area to [¹⁴C] Chrysamine Gbinding in the cerebellum (CB) of the same brain. Horizontal barsrepresent the mean and error bars represent the standard error forcontrol (circles), and AD brain (diamonds in 9A and 9B). Brain areasinclude the frontal pole (FP), head of caudate (CAU), superior/middlefrontal (SMF), superior temporal (ST), inferior parietal (IP), andoccipital (OC) cortex. Asterisks indicate significant differencescompared to control (*p<0.05; **p<0.001). Two Down's syndrome brainsamples are indicated in FIG. 9C. The diamonds in 9C represent a brainfrom a 23 year old Down's syndrome patient not yet symptomatic with AD.The triangles in 9C represent a 51 year old Down's syndrome patient whohad developed AD as do the vast majority of Down's syndrome patients bytheir 40's.

FIG. 10 is a graph illustrating the tissue levels of Chrysamine G inmice injected with [¹⁴C] Chrysamine G in the lateral tail vein andsacrificed at the times indicated. The open symbols and thin linesrepresent absolute radioactivity in units of cpm/g tissue (left axis).The closed symbols and solid lines represent the ratio of brainradioactivity to that in kidney (top) or blood (middle). The ratios areplotted on the right axis.

FIG. 11. TOP: Section from the inferior temporal lobe of AD brainstained with 1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene by themethod of Stokes and Trickey, J. Clin. Pathol. 26: 241-242 (1973).Visible are a large number of neuritic plaques, a neurofibrillary tangleand frequent neuropil threads. Cerebrovascular amyloid also is intenselystained (not shown). The photomicrograph was obtained using fluorescencemicroscopy. BOTTOM: Section of transgenic mouse brain[Tg(HuAPP695.SWE)2576; Hsiao et al. Science 274: 99-102 (1996)]similarly stained with 1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzeneshowing an intensely stained plaque.

FIG. 12. A bar graph showing the effect of increasing concentrations ofAβ(25-35) in the presence and absence of Chrysamine G on the cellularredox activity of rat pheochromocytoma (PC-12) cells as measured by3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT,reduction. The reduction product of MTT absorbs at 560 nm which isplotted on the vertical axis. The effect of Aβ(25-35) alone is shown inthe filled bars and shows a dose dependent decrease in MTT reduction.Significant differences from control (no Aβ(25-35), no Chrysamine G) areshown in white inside the filled bars. The protective effect of 20 μMChrysamine G is shown in the open bars. Significant differences betweenMTT reduction in the presence and absence of Chrysamine G are shown inblack inside the open bars.

FIG. 13. A bar graph showing the protective effect of increasingconcentrations of Chrysamine G against the Aμ(25-35)-induced reductionof cellular redox activity of rat pheochromocytoma (PC-12) cells asmeasured by 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, MTT, reduction. The reduction product of MTT absorbs at 560 nmwhich is plotted on the vertical axis. The effect of Chrysamine G in theabsence of Aβ(25-35) is shown in the filled bars. There was nosignificant difference between control (no Aβ(25-35), no Chrysamine G)and any of the concentrations of Chrysamine G in the absence ofAβ(25-35). MTT reduction in the presence of 1 μM Aβ(25-35) andincreasing concentrations of Chrysamine G is shown in the open bars.Significant differences in MTT reduction between the presence andabsence of Aβ(25-35) at each concentration of Chrysamine G are shown inwhite inside the filled bars. Significant differences in MTT reductionbetween the Aβ(25-35) control (no Chrysamine G) and Aβ(25-35) plusincreasing concentrations of Chrysamine G are shown in black inside theopen bars.

FIG. 14. Comparison of the effects of Chrysamine-G and the inactivephenol derivative on the toxicity induced by Aβ(25-35). 1 μM Aβ(25-35)was present in all experiments except control. Chrysamine-G showedprotective effects at 0.1 and 1 μM, but the phenol derivative showed noprotective effects, and perhaps enhanced the toxicity of Aβ.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention exploits the ability of alkyl, alkenyl and alkynylChrysamine G derivatives and radiolabeled derivatives thereof to crossthe blood brain barrier in vivo and bind to Aβ deposited in plaques, toAβ deposited in cerebrovascular amyloid, and to the amyloid consistingof the protein deposited in NFT. Chrysamine G is a Congo red derivativewith the key structural difference being that the sulfonic acid moietiesfound in Congo red are replaced by carboxylic acid groups in ChrysamineG (FIG. 1). This structural alteration allows Chrysamine G to enter thebrain better than Congo red and large macromolecules such as antibodies.Tubis et al., J. Am. Pharmaceut. Assn. 49: 422 (1960). Also, ChrysamineG may be a more specific marker for AD pathophysiology than antibodies,which would also label non-fibrillar Aβ deposits of uncertainpathological significance.

The alkyl, alkenyl and alkynyl Chrysamine G derivatives of the presentinvention have each of the following characteristics: (1) specificbinding to synthetic Aβ in vitro, (2) binding to β-sheet fibril depositsin brain sections (3) ability to cross a non-compromised blood brainbarrier in vivo.

The method of this invention determines the presence and location ofamyloid deposits in an organ or body area, preferably brain, of apatient. The present method comprises administration of a detectablequantity of a pharmaceutical composition containing a compound ofFormula I, as defined above, called a “detectable compound,” or apharmaceutically acceptable water-soluble salt thereof, to a patient. A“detectable quantity” means that the amount of the detectable compoundthat is administered is sufficient to enable detection of binding of thecompound to amyloid. An “imaging effective quantity” means that theamount of the detectable compound that is administered is sufficient toenable imaging of binding of the compound to amyloid.

The invention employs amyloid probes which, in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), or gamma imaging such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT), are used to quantify amyloid deposition in vivo. The term “invivo imaging” refers to any method which permits the detection of alabeled compound of Formula I, as described above, of the presentinvention. For gamma imaging, the radiation emitted from the organ orarea being examined is measured and expressed either as total binding oras a ratio in which total binding in one tissue is normalized to (forexample, divided by) the total binding in another tissue of the samesubject during the same in vivo imaging procedure. Total binding in vivois defined as the entire signal detected in a tissue by an in vivoimaging technique without the need for correction by a second injectionof an identical quantity of labeled compound along with a large excessof unlabeled, but otherwise chemically identical compound. A “subject”is a mammal, preferably a human, and most preferably a human suspectedof having dementia.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,radioactive isotopes and ¹⁹F are particularly suitable for in vivoimaging in the methods of the present invention. The type of instrumentused will guide the selection of the radionuclide or stable isotope. Forinstance, the radionuclide chosen must have a type of decay detectableby a given type of instrument. Another consideration relates to thehalf-life of the radionuclide. The half-life should be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that the host does not sustain deleteriousradiation. The radiolabeled compounds of the invention can be detectedusing gamma imaging wherein emitted gamma irradiation of the appropriatewavelength is detected. Methods of gamma imaging include, but are notlimited to, SPECT and PET. Preferably, for SPECT detection, the chosenradiolabel will lack a particulate emission, but will produce a largenumber of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁹F whichwill annihilate to form two 511 keV gamma rays which will be detected bythe PET camera.

In the present invention, amyloid binding compounds/probes are madewhich are useful for in vivo imaging and quantification of amyloiddeposition. These compounds are to be used in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), positron emission tomography (PET),and single-photon emission computed tomography (SPECT). In accordancewith this invention, the alkyl, alkenyl and alkynyl Chrysamine Gderivatives may be labeled with ¹⁹F or ¹³C for MRS/MRI by generalorganic chemistry techniques known to the art. See, e.g., March, J.“ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rdEdition, 1985), the contents of which are hereby incorporated byreference. The alkyl, alkenyl and alkynyl Chrysamine G derivatives alsomay be radiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniqueswell known in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, N.Y. 1986) thecontents of which are hereby incorporated by reference. The alkyl,alkenyl and alkynyl Chrysamine G derivatives also may be radiolabeledwith 1231 for SPECT by any of several techniques known to the art. See,e.g., Kulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), thecontents of which are hereby incorporated by reference. In addition, thealkyl, alkenyl and alkynyl Chrysamine G derivatives may be labeled withany suitable radioactive iodine isotope, such as, but not limited to¹³¹I, ¹²⁵I, or ¹²³I, by iodination of a diazotized amino derivativedirectly via a diazonium iodide, see Greenbaum, F. Am. J. Pharm. 108: 17(1936), or by conversion of the unstable diazotized amine to the stabletriazene, or by conversion of a non-radioactive halogenated precursor toa stable tri-alkyl tin derivative which then can be converted to theiodo compound by several methods well known to the art. See, Satyamurthyand Barrio J. Org. Chem. 48: 4394 (1983), Goodman et al., J. Org. Chem.49: 2322 (1984), and Mathis et al., J. Labell. Comp. and Radiopharm.1994: 905; Chumpradit et al., J. Med. Chem. 34: 877 (1991); Zhuang etal., J. Med. Chem. 37: 1406 (1994); Chumpradit et al., J. Med. Chem. 37:4245 (1994). For example, a stable triazene or tri-alkyl tin derivativeof alkyl, alkenyl and alkynyl Chrysamine G derivatives is reacted with ahalogenating agent containing ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹F.Thus, the stable triazene and tri-alkyl tin derivatives of Chrysamine Gand its analogues are novel precursors useful for the synthesis of manyof the radiolabeled compounds within the present invention. As such,these triazene and tri-alkyl tin derivatives are one embodiment of thisinvention.

The alkyl, alkenyl and alkynyl Chrysamine G derivatives also may beradiolabeled with known metal radiolabels, such as Technetium-99 m(^(99 m)Tc). Modification of the substituents to introduce ligands thatbind such metal ions can be effected without undue experimentation byone of ordinary skill in the radiolabeling art. The metal radiolabeledalkyl, alkenyl and alkynyl Chrysamine G derivative can then be used todetect amyloid deposits.

The methods of the present invention may use isotopes detectable bynuclear magnetic resonance spectroscopy for purposes of in vivo imagingand spectroscopy. Elements particularly useful in magnetic resonancespectroscopy include ¹⁹F and ¹³C.

Suitable radioisotopes for purposes of this invention includebeta-emitters, gamma-emitters, positron-emitters, and x-ray emitters.These radioisotopes include ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br, and ⁷⁶Br.Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) orSpectroscopy (MRS), according to this invention, include ¹⁹F and ¹³C.Suitable radioisotopes for in vitro quantification of amyloid inhomogenates of biopsy or post-mortem tissue include ¹²⁵I, ¹⁴C, and ³H.The preferred radiolabels are ⁸F for use in PET in vivo imaging, ¹²³ foruse in SPECT imaging, ¹⁹F for MRS/MRI, and ³H or ¹⁴C for in vitrostudies. However, any conventional method for visualizing diagnosticprobes can be utilized in accordance with this invention.

The method could be used to diagnose AD in mild or clinically confusingcases. This technique would also allow longitudinal studies of amyloiddeposition in human populations at high risk for amyloid deposition suchas Down's syndrome, familial AD, and homozygotes for the apolipoproteinE4 allele. Corder et al., Science 261: 921 (1993). A method that allowsthe temporal sequence of amyloid deposition to be followed can determineif deposition occurs long before dementia begins or if deposition isunrelated to dementia. This method can be used to monitor theeffectiveness of therapies targeted at preventing amyloid deposition.

Generally, the dosage of the detectably labeled alkyl, alkenyl andalkynyl Chrysamine G derivative will vary depending on considerationssuch as age, condition, sex, and extent of disease in the patient,contraindications, if any, concomitant therapies and other variables, tobe adjusted by a physician skilled in the art. Dosage can vary from0.001 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 100 mg/kg.

Administration to the subject may be local or systemic and accomplishedintravenously, intraarterially, intrathecally (via the spinal fluid) orthe like. Administration may also be intradermal or intracavitary,depending upon the body site under examination. After a sufficient timehas elapsed for the compound to bind with the amyloid, for example 30minutes to 48 hours, the area of the subject under investigation isexamined by routine imaging techniques such as MRS/MRI, SPECT, planarscintillation imaging, PET, and emerging imaging techniques, as well.The exact protocol will necessarily vary depending upon factors specificto the patient, as noted above, and depending upon the body site underexamination, method of administration and type of label used; thedetermination of specific procedures would be routine to the skilledartisan. For brain imaging, preferably, the amount (total or specificbinding) of the bound radioactively labelled Chrysamine G or ChrysamineG derivative or analogue is measured and compared (as a ratio) with theamount of labelled Chrysamine G or Chrysamine G derivative bound to thecerebellum of the patient. This ratio is then compared to the same ratioin age-matched normal brain.

The pharmaceutical compositions of the present invention areadvantageously administered in the form of injectable compositions. Atypical composition for such purpose comprises a pharmaceuticallyacceptable carrier. For instance, the composition may contain about 10mg of human serum albumin and from about 0.5 to 500 micrograms of thelabeled alkyl, alkenyl and alkynyl Chrysamine G derivative permilliliter of phosphate buffer containing NaCl. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: MackPublishing Co. pp. 1405-1412 and 1461-1487 (1975) and THE NATIONALFORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association(1975), the contents of which are hereby incorporated by reference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobials, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components of the pharmaceutical composition are adjustedaccording to routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

Particularly preferred pharmaceutical compositions of the presentinvention are those that, in addition to specifically binding amyloid invivo and capable of crossing the blood brain barrier, are also non-toxicat appropriate dosage levels and have a satisfactory duration of effect.

Molecular Modeling

Molecular modeling was done on an Evans and Sutherland PS-330 computergraphics system, running the computer modeling program MacroModel(Version 2.5 available from C. Still at Columbia University) to generatethe Aβ peptide chains in the anti-parallel beta-sheet conformation.Kirschner et al., Proc. Natl. Acad. Sci. U.S.A. 83: 503 (1986). Theamyloid peptides were used without further structural refinement. The Aβpeptides were aligned so that alternate chains were spaced 4.76 Å apart,characteristic of beta-sheet fibrils. Kirschner, supra. Chrysamine G wasenergy minimized and aligned with the fibril model to maximize contactwith lysine-16 of Aβ(10-43) and the hydrophobic phenylalanine-19 and -20region.

Characterization of Specific Binding to Aβ Synthetic Peptide: Affinity,Kinetics, Maximum Binding

The characteristics of Chrysamine G and Chrysamine G derivative bindingis first analyzed using synthetic Aβ peptide called Aβ(10-43). The 10-43peptide was chosen because it has been shown that this peptide providesa model system containing all of the characteristic structural featuresof Aβ peptides. Hilbich et al., J. Mol. Biol. 218: 149 (1991). The 10-43amino acid fragment of Aβ was synthesized with 9-fluorenylmethylchloroformate (FMOC) chemistry by the Peptide Synthesis Facility of theUniversity of Pittsburgh. The peptide was characterized by massspectrometry and the major component had an M_(R) of 3600 g/mole (calc.3598). The peptide was further purified by the method of Hilbich et al.which, in brief, consisted of sequential size-exclusion chromatographyon a Biogel P10 column (2×180 cm, 200-400 mesh, Biorad, Richmond,Calif.) in 70% formic acid followed by a second elution through a BiogelP4 column (2×180 cm, 200-400 mesh) in 1M acetic acid. Hilbich et al., J.Mol. Biol. 218: 149 (1991). The peptide was lyophilized and stored at−80° C. until used in the binding assays.

Amino acid sequence for Aβ(10-43) is as follows 10 11 12 13 14 15 16 1718 19 20 21 Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala 22 23 24 2526 27 28 29 30 31 32 33 Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly34 35 36 37 38 39 40 41 42 43 Leu Met Val Gly Gly Val Val Ile Ala Thr

Binding assay to synthetic Aβ(10-43)

Binding assays were performed in 12×75 mm borosilicate glass tubes.Various concentrations of nonradioactive Chrysamine G derivatives wereadded in 10% ethanol/water. Ethanol was necessary to prevent the micelleformation which occurs with these diazo dye derivatives, since themicelles are trapped by the filter even in the absence of peptide. Tothe above solution, 25 μl of a 0.36 mg/ml suspension of Aβ(10-43) in H₂Owas added and 10% ethanol was added to bring the volume to 950 μl. Afterincubating for 10 min at room temperature, 50 μl of [¹⁴C] Chrysamine Gin 40% ethanol was added, resulting in a final concentration of [¹⁴C]Chrysamine G of 100-125 nM depending on the preparation of [¹⁴C]Chrysamine G used. The binding mixture was incubated for 30 min at roomtemperature. Bound and free radioactivity were separated by vacuumfiltration through Whatman GF/B filters using a Brandel M-24R CellHarvester (Gaithersburg, Md.) followed by two 3-ml washes with 10%Filters were equilibrated overnight in 4 ml Cytoscint®-ES scintillant(ICN Biochemicals, Inc., Irvine, Calif. in 7.0 ml plastic scintillationvials before counting. In this and all binding assays, incubations weredone at least in triplicate and the results expressed as mean ± standarddeviation.

Kinetic Studies

Kinetics studies of [¹⁴C] Chrysamine G binding to Aβ(10-43) wereperformed in ×100 mm borosilicate glass tubes by the filtration assaydescribed above. For the kinetics of association, 25 μl of 0.36 mg/mlAβ(10-43) were placed in 475 μl of 10% ethanol and 4.5 ml of 125 nM[¹⁴C] Chrysamine G was added to the solution at time zero. The mixturewas rapidly vortexed and the binding reaction was stopped by vacuumfiltration through Whatman GF/B filters using a Brandel M-24R CellHarvester (Gaithersburg, MD) followed by two 3-ml washes with 10%ethanol at room temperature at times of 5, 10, 20, 30, 45, 60, 75, 135,240, and 300 sec.; bound radioactivity was determined as above.

For the kinetics of dissociation, 25 μl of 0.36 mg/ml Aβ(10-43) wereplaced in 450 μl of 10% ethanol followed by 25 μl of 2.5 μM [¹⁴C]Chrysamine G in 40% ethanol. This mixture was vortexed and incubated for30 min at room temperature. The mixture was diluted with 4.5 ml of 10 μMnonradioactive Chrysamine G in 10% ethanol at time zero, the mixture wasrapidly vortexed, and the dissociation was stopped by filtration asabove at times of 0.5, 1.5, 3, 5, and 15 min, and bound radioactivitywas determined as above.

Characterization of Specific Binding to Alzheimer's Disease Brain

Binding of Chrysamine G to AD and Control Brain Homogenates

Autopsy brain samples were obtained from the Neuropathology Core of theAlzheimer's Disease Research Center of the University of Pittsburgh.Controls were defined as not meeting neuropathological criteria for AD(sufficient number of NPs or NFTs) according to the standards specifiedin a published NIA conference report. Khachaturian, Arch. Neurol. 42:1097 (1985). Brain samples from eight control (ages 58-75), eleven AD(ages 61-84), and two Down syndrome brains (ages 23 and 51) werestudied. There were six high-plaque (>20 NPs/×200 magnification) andfive low-plaque (<20 NPs/×200 magnification) AD brains. Two controlswere clinically demented but had no NPs or NFTs and received thediagnosis of “Dementia Lacking Distinctive Histology”. Knopman Dementia4: 132 (1993). Another control had dementia and olivopontocerebellaratrophy. The other controls had no clinical or histological evidence ofneurologic disease. Autopsy samples were immediately frozen at −70° C.and stored at that temperature until homogenized. The numbers of NPs andNFTs were counted in sections of five separate but adjacent fields (×200magnification) between cortical layers 2 and 4 in the cortex at thejunction of superior and middle frontal gyri and superior temporalisocortex of all brains studied. A qualitative assessment of thepresence of amyloid angiopathy in the superior/middle frontal cortex wasmade. The Bielschowsky silver impregnation method was used to identifyNPs and NFTs and Congo red staining was used to identify cerebralamyloid angiopathy. Details of this procedure have been previouslypublished. Moossy et al., Arch. Neurol. 45: 251 (1988). Samples used forCG binding to the superior/middle frontal or superior temporal cortexwere adjacent on gross dissection to those used for NP and NFT counts.

Approximately 100 mg of tissue from the junction of the superior andmiddle frontal cortex, superior temporal cortex, frontal pole, head ofthe caudate, inferior parietal cortex, occipital cortex, or cerebellumwere homogenized with a Polytron® tissue homogenizer (PT 10/35, BrinkmanInstruments Inc., Westbury, N.Y.) for 30 sec at setting 6 in 10% ethanolat a concentration of 10-20 mg brain/ml. Not all areas were availablefrom each brain. Aliquots of 25-150 μl tissue (about 25-300 μg ofprotein by the method of Lowry et al., J. Biol, Chem. 193: 265 (1951))were incubated in 12×75 mm borosilicate glass tubes at room temperaturewith 10-750 nM [¹⁴C]CG (26.8 Ci/mole) in a final volume of 1.0 ml of 10%ethanol for 30 min at room temperature. The standard conditions employedabout 150 μg of protein and 75 nM [¹⁴C]CG for the cerebellar ratiostudies and about 75 μg of protein and 150 nM [¹⁴C]CG for thecorrelative studies with NPs, NFTs, and amyloid angiopathy. Ethanol wasnecessary to prevent the micelle formation which occurs with diazo dyederivatives, since the micelles are trapped by the filter even in theabsence of tissue. Bound and free radioactivity were separated by vacuumfiltration through Whatman GF/B filters using a Brandel M-24R CellHarvester (Gaithersburg, Md.) followed by two 3-ml washes with 10%ethanol at room temperature. Filters were equilibrated overnight in 4 mlCytoscint®-ES scintillant (ICN Biomedicals, Inc., Irvine, Calif.) in 7.0ml plastic scintillation vials before counting. Saturable (specific)binding was defined as total binding minus residual (non-saturable)binding in the presence of 20 μM unlabelled CG. In all binding assays,incubations were done at least in triplicate and the results expressedas mean±standard error unless otherwise specified. Results wereexpressed either in absolute terms of fmol [¹⁴C]CG bound/μg protein in agiven brain area, or as a ratio of the fmol/μg protein in that brainarea to the fmol/μg protein in the cerebellum of the same brain.

Octanol/Water Partitioning

Approximately 75 μM solutions of Chrysamine G or its analogues wereprepared in 5.0 ml 1-octanol. Five ml of phosphate buffered saline (0.15M NaCl, 5 mM potassium phosphate, pH 7.4) were added and the layersmixed by rapid vortexing. The mixture was then centrifuged at 1,000 g tofacilitate the formation of two clear phases. The layers were separatedusing a separatory funnel and 600 μl of each layer was diluted with 400μl of ethanol and the absorbance measured at 389 nm for Chrysamine G orthe λ_(max) for each analogue. Concentrations were determined aftercorrection for the molar absorptivity differences in the two solventsand the partition coefficient expressed as the concentration in theoctanol layer divided by the concentration in the aqueous layer.Experiments were done in triplicate.

Imaging the Binding of Chrysamine G to Amyloid Deposits in Alzheimer'sDisease Brain

For visual demonstration of CG derivative binding to tissue, 8 micronparaffin sections of an AD brain with heavy deposits of cerebrovascularamyloid were stained with1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene by a modification ofthe method of Stokes and Trickey, J. Clin. Pathol. 26: 241-242 (1973)with 1 mM 1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene substitutedfor Congo red, but the procedure was otherwise identical. Stained slideswere examined using fluorescence microscopy.

Determining Compound's Ability to Cross the Blood Brain Barrier

Mouse studies Female Swiss-Webster mice were injected in the lateraltail vein with approximately 0.03 μCi/g of [¹⁴C] Chrysamine G in a 0.9%NaCl solution. Mice were sacrificed by cervical dislocation at intervalsof 15 min, 35 min, 1 hr, 4 hr, and 24 hr after injection. The carotidblood, brain, liver, and kidneys were rapidly obtained, weighed, andhomogenized in distilled/deionized H₂O using a ground glass homogenizer.An aliquot was weighed into an 18.0 ml plastic scintillation vial(Beckman Poly-Q-Vial) and counted after addition of 10.0 ml ofscintillation cocktail (Cytoscint®-ES (ICN)) and overnightequilibration. The [¹⁴C] Chrysamine G content of the tissues wasexpressed as cpm/mg tissue.

Experiments in which radioactivity was extracted from tissues wereperformed as above except 0.05 μCi/g of [¹⁴C] Chrysamine G was injectedand the mice were sacrificed at 60 min. Brain and liver were thenremoved and extracted with a Folch procedure. Folch et al., J. Biol.Chem. 226: 447 (1957). In both tissues, over 95% of the extractedradioactivity was contained in the organic layer. The organic layer wasevaporated to dryness, resuspended in a minimal amount of 10%methanol/90% ACN, and injected onto a silica column (Prep Nova Pak HRSilica, 7.8×300 mm, Waters, Milford, Mass.) and eluted isocraticallywith the same solvent. Under these conditions, 99% of the radioactivityis eluted in the solvent front, but most lipids are retained longer,making the fraction eluting in the solvent front suitable for injectiononto the reverse-phase C4 column system described above. The entiresolvent front was collected, dried, and resuspended in 10% ACN/90%sodium phosphate buffer (5mM, pH 6) and injected, along with authenticnon-radioactive Chrysamine G, onto the C4 column. One minute fractionswere collected and counted after addition of 10 ml of Cytoscint®-ES.

In yet another embodiment, the invention relates to a pharmaceuticalcomposition and method for preventing cell degeneration and toxicityassociated with fibril formation in certain “amyloidosis associated”conditions such as Alzheimer's Disease, Down's Syndrome and Type 2diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch),amyloid A (reactive), secondary amyloidosis, familial mediterraneanfever, familial amyloid nephropathy with urticaria and deafness(Muckle-wells Syndrome), amyloid lambda L-chain or amyloid kappa L-chain(idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, (systemic senile amyloidosises), AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy - Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele. This method involves administering apharmaceutical composition comprising Chrysamine G, or one of the abovedescribed derivatives thereof, to a subject suspected of having or athigh risk of developing such amyloidosis associated condition.

Because certain diazo compounds could be carcinogenic, the therapeuticcompounds of the present invention include only non-toxic,non-carcinogenic compounds. That is, the present invention addresses theproblems with potential carcinogenicity by using only alkyl, alkenyl andalkynyl compounds which do not contain groups that could be metabolizedto carcinogenic benzidine compounds.

Any potential problems with lower bioavailability also is avoided by theuse of alkyl, alkenyl and alkynyl derivatives of the azo compounds.These compounds are not substrates for reduction by bacterial ormammalian azo reductases.

Indeed, compounds of the present invention intended for therapeutic useare advantageous over existing compounds because they contain an1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene linkage which is not asubstrate for bacterial azo-reductases in the intestines.

In vitro studies have shown that Aβ neurotoxicity requires fibrilformation and is inhibited by Congo red. Specifically, it has been shownthat the amyloid fibril-binding dye Congo red inhibits fibrillar Aβneurotoxicity by inhibiting fibril formation or by binding preformedfibrils. Lorenzo et al., Proc. Natl. Acad. Sci. USA 91: 12243-12247(1994). Congo red also has been shown to inhibit pancreatic islet celltoxicity of diabetes-associated amylin, another type of amyloid fibril.Lorenzo et al., supra. See also, Burgevin et al. NeuroReport 5: 2429(1994); Pollack et al., J. Neurosci. Letters 184: 113-116 (1995);Pollack et al. Neuroscience Letters 197: 211 (1995). These data indicatethat amyloid-binding compounds such as alkyl, alkenyl and alkynylderivatives of Chrysamine G, which are similar to Congo red but which,unlike Congo red, enter the brain well, would be effective in preventingcell degeneration and toxicity associated with fibril formation inamyloidosis associated conditions.

In Example 8 and FIGS. 12 and 13, it is shown that Chrysamine G haseffects very similar to those previously reported for Congo red inhaving a dose-dependent, protective effect in rat pheochromocytoma.Therefore, these in vitro assays provide a means for selecting compoundsfor use in pharmaceutical compositions for the prevention of celldegeneration and toxicity associated with fibril formation.

Compounds such as Chrysamine G and the above described derivativesthereof, are tested pursuant to the present invention, for in vivoefficacy in preventing amyloid fibril formation or associated cellulardegeneration, as measured by the formation of dystrophic neurites,synapse loss, neurofibrillary tangle formation and gliosis, in an animalmodel, such as the “senile animal” model for cerebral amyloidosis,Wisniewski et al., J. Neuropathol. & Exp. Neurol. 32: 566 (1973), themouse model of familial Mediterranean fever (Neurochem., Inc. Kingston,Ontario, Canada) and the transgenic mouse model of Alzheimer-typeneuropathology, Games et al., Nature 373: 523-527 (1995); Hsiao et al.Science 274: 99-102 (1996). In the familial Mediterranean fever model,the animals develop systemic amyloidosis. In an in vivo assay accordingto this invention, serial necropsies in animals treated and untreatedwith the compounds of the present invention to evaluate the inhibitionof amyloid formation are compared. In the animal models for cerebralamyloid formation, in addition to following amyloid formation serially,the presence of amyloid-associated neurodegeneration, as measured by theformation of dystrophic neurites, synapse loss, neurofibrillary tangleformation and gliosis, also is assessed in serial necropsies in animalstreated and untreated with the compounds of the present invention.

According to the present invention, a pharmaceutical compositioncomprising Chrysamine G or derivatives thereof, is administered tosubjects in whom amyloid or amyloid fibril formation, cell degenerationand toxicity are anticipated. In the preferred embodiment, such subjectis a human and includes, for instance, those who are at risk ofdeveloping cerebral amyloid, including the elderly, nondementedpopulation and patients having amyloidosis associated diseases and Type2 diabetes mellitus. The term “preventing” is intended to include theamelioration of cell degeneration and toxicity associated with fibrilformation. By “amelioration” is meant the prevention of more severeforms of cell degeneration and toxicity in patients already manifestingsigns of toxicity, such as dementia.

The pharmaceutical composition for purposes of preventing celldegeneration and toxicity associated with fibril formation inamyloidosis associated diseases comprises Chrysamine G or a derivativethereof described above and a pharmaceutically acceptable carrier. Inone embodiment, such pharmaceutical composition comprises serum albumin,Chrysamine G or Chrysamine G derivative and a phosphate buffercontaining NaCl. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like, as described, for instance, in REMINGTON'SPHARMACEUTICAL SCIENCES, 15th Ed., Easton: Mack Publishing Co., pp.1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed.Washington: American Pharmaceutical Association (1975), and the UNITEDSTATES PHARMACOPEIA XVIII. 18th Ed. Washington: American PharmaceuticalAssociation (1995), the contents of which are hereby incorporated byreference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

According to the invention, such pharmaceutical composition could beadministered orally, in the form of a liquid or solid, or injectedintravenously or intramuscularly, in the form of a suspension orsolution. By the term “pharmaceutically effective amount” is meant anamount that prevents cell degeneration and toxicity associated withfibril formation. Such amount would necessarily vary depending upon theage, weight and condition of the patient and would be adjusted by thoseof ordinary skill in the art according to well-known protocols. In oneembodiment, a dosage would be between 0.1 and 100 mg/kg per day, ordivided into smaller dosages to be administered two to four times perday. Such a regimen would be continued on a daily basis for the life ofthe patient. Alternatively, the pharmaceutical composition could beadministered intramuscularly in doses of 0.1 to 100 mg/kg every one tosix weeks.

In yet another embodiment, the invention relates to a method ofdetecting amyloid deposits in biopsy or post-mortem tissue. The methodinvolves incubating formalin-fixed tissue with a solution of a compoundof Formula I, described above. Preferably, the solution is 25-100%ethanol, (with the remainder being water) saturated with the compound ofFormula I. Upon incubation, the compound stains or labels the amyloiddeposit in the tissue, and the stained or labelled deposit can bedetected or visualized by any standard method. Such detection meansinclude microscopic techniques such as bright-field, fluorescence,laser-confocal and cross-polarization microscopy.

In yet another embodiment, the invention relates to a method ofquantifying the amount of amyloid in biopsy or post-mortem tissue. Thismethod involves incubating a labelled alkyl, alkenyl and alkynylderivative of Chrysamine G, preferably the compounds of Formula I, or awater-soluble, non-toxic salt thereof, with homogenate of biopsy orpost-mortem tissue. The tissue is obtained and homogenized by methodswell known in the art. The preferred label is a radiolabel, althoughother labels such as enzymes, chemiluminescent and immunofluorescentcompounds are well known to skilled artisans. The preferred radiolabelis ¹²⁵I, ₁₄C or 3H, the preferred label substituent of Formula I is atleast one of R₁-R₇, R₁₀-R₂₇. Tissue containing amyloid deposits willbind to the labeled alkyl, alkenyl and alkynyl derivatives of ChrysamineG. The bound tissue is then separated from the unbound tissue by anymechanism known to the skilled artisan, such as filtering. The boundtissue can then be quantified through any means known to the skilledartisan. See Example 3. The units of tissue-bound radiolabeledChrysamine G derivative are then converted to units of micrograms ofamyloid per 100 mg of tissue by comparison to a standard curve generatedby incubating known amounts of amyloid with the radiolabeled ChrysamineG derivative.

In yet another embodiment, the invention relates to a method ofdistinguishing an Alzheimer's diseased brain from a normal braininvolving obtaining tissue from (i) the cerebellum and (ii) another areaof the same brain, other than the cerebellum, from normal subjects andfrom subjects suspected of having Alzheimer's disease. See Example 3.Such tissues are made into separate homogenates using methods well knownto the skilled artisan, and then are incubated with a radiolabeledalkyl, alkenyl and alkynyl Chrysamine G derivative. The amount of tissuewhich binds to the radiolabeled alkyl, alkenyl and alkynyl Chrysamine Gderivative is then calculated for each tissue type (e.g. cerebellum,non-cerebellum, normal, abnormal) and the ratio for the binding ofnon-cerebellum to cerebellum tissue is calculated for tissue from normaland for tissue from patients suspected of having Alzheimer's disease.These ratios are then compared. If the ratio from the brain suspected ofhaving Alzheimer's disease is above 90% of the ratios obtained fromnormal brains, the diagnosis of Alzheimer's disease is made.

EXAMPLE 1

The Synthesis of Chrysamine G and Derivatives Thereof

Synthesis of Chrysamine G

The synthesis of Chrysamine G (i.e.,4,4′-bis(3-carboxy-4-hydroxyphenylazo)-biphenyl) requires the followingreaction steps. These reaction steps will be referred to as the“Chrysamine G Synthesis” general procedure. Benzidine.2HCl (28.9 mg,0.11 mmole, Sigma Chemical Company, St. Louis, Mo.) was added to 1.5 mlof 1:1 DMSO:distilled/deionized H₂O in a 50cc round bottom flask. Eachof the reaction steps were carried out at 0° C. unless otherwisespecified. Twenty-nine μl of concentrated HCl were added, resulting in aclear solution after stirring. To the benzidine solution, a solution of15.5 mg (0.22 mmole) of NaNO₂ in 300 μl of 1:1 DMSO/H₂O was addeddrop-wise, resulting in a pH of about 2-3. The reaction mixture wasstirred for 45 min, and then to this tetra-azotized benzidine mixturewas added drop-wise over a 10 min period to 24.8 mg (0.18 mmole) ofmethyl salicylate (Aldrich) dissolved in 2.0 ml of 100% DMSO containing250 mg/ml Na₂CO₃ in suspension, keeping the pH about 10.5. The resultingmixture was stirred for 1 hr at 0° C., and then overnight at roomtemperature.

After this time, the pH was adjusted to about 7 and the mixture wasextracted with three 50 ml portions of chloroform. The combinedchloroform extracts were washed with three 50 ml portions of H₂O, andthen taken to dryness yielding the dimethyl ester of Chrysamine G (i.e.,4,4′-bis (3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl), which wasfurther purified by recrystallization from chloroform/hexane. The esterwas then hydrolysed by dissolution in about 100 ml of 1:1 ethanol:H₂Ocontaining four equivalents of NaOH and refluxed for three hours.Evaporation of the ethanol followed by lyophilization of the H₂O yieldedthe tetra-sodium salt of Chrysamine G. The free acid of Chrysamine G wasformed by dissolving the tetra-sodium salt in H₂O, washing once withchloroform to remove any unhydrolysed dimethyl ester, lowering the pH toabout 2 and extracting with three 50 ml portions of ethyl acetate. Thecombined ethyl acetate extracts were washed with three 50 ml portions ofH₂O and taken to dryness.

Under these conditions, there was no remaining methyl salicylate,salicylic acid, or benzidine, and only trace amounts of themono-substituted product,4-hydroxy-4′-(3-carboxy-4-hydroxyphenylazo)-biphenyl by reverse-phaseHPLC using a C4 column (Vydac 214-TP510) using a solvent system ofsodium phosphate buffer (5 mM, pH 6):acetonitrile (ACN) 90:10,isocratically, for 10 min and then increased to 500% ACN over the next20 min at a flow rate of 3.5 ml/min. The column eluant was monitored at290 and 365 nm with a dual wavelength, diode array detector (PerkinElmer 235C). Under these conditions, Chrysamine G eluted at 17.6 min.

The structure of Chrysamine G and derivatives was confirmed by protonNMR at 500 MHz in DMSO-d₆ with TMS as the internal standard. The peakassignments for the tetra-sodium salt of Chrysamine G were as followswith SA referring to protons at the specified ring position on thesalicylic acid moiety and BZ referring to protons on the benzidinemoiety: SA-3, doublet J=8.73 Hz at 6.75 parts per million (ppm); SA-4,doublet of doublets J=8.73 and 2.72 Hz at 7.82 ppm; BZ-2/6, doubletJ=8.44 Hz at 7.91 ppm; BZ-3/5, doublet J-8.44 Hz at 7.95 ppm; and SA-6,doublet J-2.72 Hz at 8.28 ppm. The UV/visible spectrum in 40% ethanolshowed a λ_(max) at 389 nm. The molar absorptivity of Chrysamine G wasdetermined by calculating the concentration of Chrysamine G throughcomparison of peak areas to an internal standard by NMR and thenimmediately running the UV/vis spectrum of an aliquot of the NMR samplediluted in 40% ethanol. The molar absorptivity in 40% ethanol at 389 nmwas 5.5×10⁴ AU/(cm·M).

[¹⁴C] Chrysamine G was synthesized by a modification of the aboveprocedure. The tetra-azotization of benzidine was performed as describedabove except in 100% H₂O. Fifty μl of 2.5 M Na₂CO₃ in H₂0 were added to50 μCi of crystalline salicylic acid-carboxy-¹⁴C (Sigma) in a 0.5 mlconical glass vial. Sixty μl of the tetra-azotized benzidine mixture wasadded to the conical vial, vortexed and kept at 0° C for 1 hr. Toprevent formation of the mono-substituted benzidine by-product product,12.5 μl of 250 mM non-radioactive salicylic acid (Sigma) in 2.5 M Na₂CO₃was added to the reaction mixture and maintained for 1 hr at 0° C. Thevial was kept overnight at room temperature. The entire mixture wasdissolved in a minimal amount of 35% ACN and injected onto the C4 columnas described above. The peak corresponding to the Chrysamine G standardwas collected and lyophilized. A specific activity of 26.8 Ci/mole wascalculated by determining the absorbance at 389 nm and then counting theradioactivity in an aliquot of the same sample. The [¹⁴C] Chrysamine Gwas stored in 40% ethanol. When the purified [¹⁴C] Chrysamine G wasre-injected onto the C4 column and eluted isocratically with 21% ACN at3.5 ml/min, >98% of the radioactivity co-eluted with authenticChrysamine G at 10.4 min. Many of the Chrysamine G derivatives weresynthesized using this “Chrysamine G Synthesis” general procedure, withthe exceptions noted below. Structures of the derivatives were verifiedby NMR. FIG. 1 shows the chemical structure of Chrysamine G and severalderivatives.

Synthesis of Alkenyl (CH═CH) derivatives of Chrysamine-G

4,4′-biphenyldicarboxylic acid (Aldrich) is converted by reduction withLiAlH₄ to 4,4′-bis(hydroxymethyl)biphenyl, which, in turn, is convertedto 4,4′-bis(iodomethyl)biphenyl by reaction with NaI and BF₃-etherate inACN. The iodo compound is heated to 90° C. for one hour with excesstriethyl phosphite to produce tetraethyl4,4′-biphenyldimethylphosphonate. Similar treatment of1,4-napthalene-dicarboxylic acid (Aldrich) or9,10-anthracene-dicarboxylic acid (Aldrich) yields the respectivetetraethyl phoshonates. After recrystallization from hexane, thephosphonate is dissolved in DMF and treated with a ten-fold excess ofsodium methoxide, followed by two equivalents of 5-formylsalycylic acidin DMF. After stirring at room temperature for 24 hrs, the reactionmixture is poured into water. Acidification of the water to pH 5.0 withHCl causes precipitation of the flourescent product,4,4′-bis(3-carboxy-4-hydroxyphenylethenyl)-biphenyl, which can beselectively extracted into ethyl acetate from any mono-substitutedby-product. Similar treatment of tetraethyl p-xylylenediphosphonate (TCIAmerica) with 5-formylsalicylic acid, or its derivatives, gives1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene. Likewise1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-naphthalene or9,10-bis(3-carboxy-4-hydroxyphenylethenyl)-anthracene is obtained bytreating the appropriate phosphonate with 5-formylsalicylic acid. Otherderivative are obtained by the use of other formylsalicylic acidcongeners, formyl benzoic acids, or hydroxy- or methoxybenzaldehydes.

When backbone linkers other than those listed above are desired, theappropriate dicarboxylic acid (such as 2-bromoterephthalic acid(Aldrich)) is reduced to the diol, converted to the iodide, and then tothe tetraethyl diphosphonate as described above. When side groups otherthan salicylic acid are desired, the appropriate phenol (which willusually also contain an acidic functionality as well) is first iodinatedortho or para (depending on the presence of other substituents) to thephenol and then formylated at the iodo-position by standard methods.

Synthesis of Alkyl-Substituted Alkenyl (CR′═CR′) derivatives ofChrysamine-G

4,4′-biphenyldicarboxylic acid (Aldrich) or 1,4-benzenedicarboxylic acid(Aldrich) is first converted by reduction with LiAlH₄ to thebis(hydroxymethyl) compound, which, in turn, is converted to thedicarboxaldehyde by treatment with BaMnO₄ in ethyl acetate. Thisdialdehyde is reacted with R′MgX (where R′ is a lower alkyl group and Xis Br or I) via the Grignard reaction to produce HOCR′H—Ph—Ph—CR′H—OH orHOCR′H—Ph—CR′H—OH. The alkyl-substituted bis(hydroxymethyl) compound isconverted to the alkyl-substituted bis(iodomethyl) compound by reactionwith NaI and BF₃-etherate in ACN. The iodo compound is heated to 90° C.for about one hour with excess triethyl phosphite to produce thealkyl-subsitiuted tetraethyl dimethylphosphonate. Similar treatment of1,4-naphthalene-dicarboxylic acid (Aldrich) or9,10-anthracene-dicarboxylic acid (Aldrich) yields the respectivealkyl-substituted tetraethyl dimethylphosphonates.

Alkyl-substituted aklenyl compounds of three varieties, namelyAR—CR′═CR′—Q, AR—CR′═CH—Q or Ar—CH═CR′—Q (where R′ is a lower alkylgroup and Q is as defined in Formula I), can then be synthesized.AR—CR′═CR′—Q compounds can be made by reacting an alkyl-substitutedtetraethyl dimethylphosphonate with a suitable aryl ketone such as5-acetylsalicyclic acid (Crescent Chemical Co., Inc., Hauppage N.Y.).AR—CR′═CH—Q compounds can be made by reacting an alkyl-substitutedtetraethyl dimethylphosphonate with a suitable aldehyde such as5-formylsalicyclic acid (Aldrich). AR—CH═CR′—Q compounds can be made byreacting tetraethyl dimethylphosphonate with a suitable aryl ketone suchas 5-acetylsalicyclic acid (Crescent Chemical Co., Inc., Hauppage N.Y.).The reaction conditions are identical to those used to make the alkenyl(CH═CH) derivatives described above.

Synthesis of Alkynyl (C≡C) Derivatives of Chrysamine G 5-Iodosalicylicacid (Aldrich Chemical Company, Milwaukee, Wis.) is converted to themethyl ester by reaction with methanol, trimethyl orthoformate andsulfuric acid. The 5-iodosalicylic acid methyl ester thus obtained isreacted with (trimethylsilyl)acetylene (Aldrich Chemical Company,Milwaukee, Wis.) in the presence of palladium. The trimethylsilyl groupis removed and two equivalents of the resultant 5-acetylenylsalicylicacid methyl ester is reacted with 4,4′-dibromobiphenyl (Aldrich ChemicalCompany, Milwaukee, Wis.) in the presence of palladium as above. Theresultant alkynyl analogue of Chrysamine G,4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylacetylenyl)-biphenyl isprepared by hydrolysis of the ester as described above.

Alternative Synthesis of Alkynyl (C≡C) and Vinyl (CH═CH) Derivatives ofChrysamine G

5-Bromosalicylic acid (Aldrich Chemical Company, Milwaukee, Wis.) isconverted to the methyl ester/methyl ether by reaction with methyliodide in the presence of K₂CO₃ as described above. The2-methoxy-5-bromobenzoic acid methyl ester thus obtained is reacted with(trimethylsilyl)acetylene (Aldrich Chemical Company, Milwaukee, Wis.) inthe presence of palladium. The trimethylsilyl group is removed and twoequivalents of the resultant 2-methoxy-5-acetylenylbenzoic acid methylester is reacted with 4,4′-dibromobiphenyl (Aldrich Chemical Company,Milwaukee, Wis.) in the presence of palladium as above. The resultantalkynyl analogue of Chrysamine G,4,4′-bis(3-carboxy-4-methoxyphenylacetylenyl)-biphenyl is prepared byhydrolysis of the ester as described above. This alkynyl analogue isreduced by conventional methods to form the vinyl analogue of ChrysamineG.

Synthesis of Alkyl (CH₂-CH₂) Derivatives of Chrysamine G

Either the alkenyl or alkynl derivatives described above arehydrogenated by standard methods using hydrogen gas and a platinum orpalladium catalyst.

Synthesis of Di-fluoro Alkenyl Chrysamine G Derivative

The 5-fluoro derivative,1,4-bis(2-hydroxy-3-carboxy-5-fluorophenylethenyl)-benzene), issynthesized by substituting 3-formyl-5-fluorosalicylic acid for5-formyl-salicylic acid. [¹⁸F]aryl fluorides derivatives of Chrysamine Gcan be prepared by substituting ¹⁸F-labeled precursors such as[¹⁸F]LiBF₄, in the Schiemann reaction, via triazene decomposition withCs [¹⁸F], or via nucleophilic ¹⁸F-for-X substitution, where X=tosyl,triflate, NO₂, ⁺N(CH₃)₃, or halogen. See Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, N.Y., 1986) andKilbourn, M. Fluorine-18 labeling of radiopharmaceuticals. (Natl. Acad.Press, Washington, D.C.) (1990).

Synthesis of Aromatic Fluoroalkyl and Fluoroalkoxy Derivatives

Aromatic fluoroalkyl derivatives are synthesized employing the method ofBishop et al., J. Med. Chem. 34: 1612 (1991) in which Claisenrearrangement of the appropriate O-allyl ethers forms an aromatic allylderivative which can be further functionalized to yield the fluoroethylor fluoropropyl derivatives. Alternatively, an aromatic iodide can bereadily converted to an aromatic alkyne consisting of two to five carbonatoms in length using the palladium-assisted coupling methodology ofSonogashira et al., Tetrahedron Letters 4467-4470 (1975). Subsequentderivatization of the alkyne yields the fluoroalkyl derivative.Fluoroalkoxy derivatives may be prepared by the method of Chumpradit etal., J. Med. Chem. 36: 21 (1993) in which alkylation of the appropriatephenol with the appropriate 1-bromo (or iodo orsulfonyloxy)-omega-fluoroalkane yields the corresponding fluoroalkoxyderivative.

Radiofluorination of Aromatic Alkylsulfonyloxy and AlkoxysulfonyloxyDerivatives

Radiofluorination to yield the aromatic [¹⁸F]fluoroalkyl and[¹⁸F]fluoroalkoxy derivatives is performed by the method of Mathis etal., Nucl. Med. Biol. 19: 571 (1992) in which aromatic alkyl- oralkoxysulfonyloxy (e.g. alkoxytosylate) derivatives are substituted with[¹⁸F]fluoride to yield aromatic [¹⁸F]fluoralkyl and [¹⁸F]fluoralkoxycompounds.

Radio-Iodination and Radio-Bromination by the Tri-Alkyl Tin Route

Synthesis of Tri-Alkyl Tin Derivatives

The general structure of the alkenyl tri-alkyl tin derivative ofChrysamine G is shown in FIG. 2B. In general, one tri-alkyl tin groupwill be substituted at the 3-position on one side of the biphenylmoiety, but other positions, including the salicylic acid orheterocyclic moiety are also potential targets. These tri-alkyl tinderivatives are stable immediate precursors for preparation of theradioiodinated and radiobrominated compounds to be used in humans. Morespecifically, these tri-alkyl tin derivatives are used to prepare thehalogenated radioactive compounds applicable for use in in vivo imagingof amyloid.

General Procedures for the Synthesis of Tri-Alkyl Tin Derivatives

Tri-alkyl tin derivatives are prepared from the appropriate arylhalides,[(C₆H₅)₃P]₃Pd(0), and hexaalkylditin by previously published proceduresincluding Kosugi, M., et al., Chem. Lett. 1981: 829; Heck, R. Pure andAppl. Chem. 1978: 691; Echavarren, A. and Stille, J. J. Am. Chem. Soc.1987: 5478; Mitchell, T. J. Organometallic Chem. 1986: 1; and Stille, J.Pure and Applied Chem. 1985: 1771. These derivatives also can beobtained by the use of n-BuLi and trialkyl tin chloride by the procedureof Mathis et al., J. Labell. Comp. and Radiopharm. 1994: 905.

Synthesis of the 3-trialkyl tin derivative of4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl

3-Bromo or3-iodo-4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl or itsdimethyl ether are prepared by synthesis of 3-bromo- or 3-iodobenzidine(see above), tetra-azotization and coupling to methyl salicylate as forthe synthesis of Chrysamine G, and methylation of the phenol asdescribed above when the methoxy compound is desired. Under an argonatmosphere, 1 mmol of the phenolic ester or the methoxy ester,[(C₆H₅)₃P]₃Pd(0) (0.1 to 0.2 mmol), hexabutylditin or hexamethyl ditin(1.25 mmol), and dioxane (25 ml) is heated at 70° C. for 16 hrs. Thereaction mixture is cooled and the solvent is evaporated. Tri-alkyl tinhalide is removed with aqueous KF. The organics are extracted with ethylacetate, dried over magnesium sulfate, filtered, and the solvent isevaporated under reduced pressure. The residue is purified on silica gelto obtain 3-trialkyltin-4,4′-bis(3-methoxycarbonyl-4-hydoxyphenylazo)-biphenyl.

Radio-Iodination or Radio-Bromination of Tri-Alkyl Tin Derivatives

The tributyl or trimethyl tin derivatives are radio-iodinated withNa[¹²⁵I ] or Na[¹²³I] or radio-brominated with Na[⁷⁵Br] or Na[⁷⁶Br] bypublished procedures such as Mathis et al., J. Labell. Comp. andRadiopharm. 1994: 905; Chumpradit et al., J. Med. Chem. 34: 877 (1991);Zhuang et al., J. Med. Chem. 37: 1406 (1994); Chumpradit et al., J. Med.Chem. 37: 4245 (1994). In general, 0.5 mg of tri-alkyl tin compound, 0.2ml of anhydrous acetonitrile, 10 μl of 2M H₃PO₄, 2-100 μl of a solutionof high specific activity (>2000 Ci/mmol) Na[¹²⁵1] or Na[¹²³I] (orNa[⁷⁵Br] or Na[⁷⁶Br]) in pH 9-12 NaOH, and dichloramine-T (DCT) (20 μlof 2.5 mg/ml DCT in acetonitrile) are placed in a 1 ml Reacti-Vial. Thevial is capped and the mixture is stirred at room temperature in thedark. The reaction is monitored by HPLC and after 30 min is quenchedwith 50 μl of 2 M Na₂S₂O₃. The product is purified by standardchromatographic techniques. Mathis et al., J. Labell. Comp. andRadiopharm. 1994: 905. Similarly, low specific activity ¹⁸F derivativesare prepared by analogous procedures.

General Procedures for the Preparation of Non-Radioactive I, Br, Cl, Fand -SH Derivatives

In general, 3- or 4-amino derivatives of 5-formylsalicylic acid, or thecorresponding derivatives of the heterocyclic analogues of salicylicacid shown in FIG. 2, are converted to the corresponding diazo compoundswith sodium nitrite and HCl or H₂SO₄. The iodine derivatives aredirectly prepared by forming the diazonium iodide which is thenconverted into the aryl iodide, or by way of the triazene intermediates.See, e.g., Greenbaum, F. Am. J. Pharm. 108: 17 (1936), Satyamurthy, N.and Barrio, J., J. Org. Chem. 48: 4394 (1983) and Goodman, M. et al., J.Org. Chem. 49: 2322 (1984). Aryl bromides and chlorides are preparedfrom the diazo compounds by treatment with CuCl or CuBr according to theSandmeyer reaction or via the triazene as for the iodine derivatives.Aryl fluorides are prepared by treating the diazonium compounds withNaBF₄, HBF₄, or NH₄BF₄ according to the Schiemann reaction or viatriazene decomposition similar to the iodine derivatives. Aryl thiolsare prepared from the diazonium compounds by treatment withsulfur-containing nucleophiles such as HS⁻, EtO-CSS⁻, and S₂ ²⁻.Alternatively, aryl thiols can be prepared by replacement of arylhalides with sulfur containing nucleophiles. These reactions aredescribed in March, J., ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS, AND STRUCTURE (3rd Edition, 1985).

General Procedures for the Preparation of Radioactive C, F and TcDerivatives

In addition to the above procedures, high specific activityradiolabeling with ^(99m)Tc for SPECT or with the positron-emittingradionuclides ¹¹C, ¹⁸F, ⁷⁵Br and ⁷⁶Br is accomplished according toliterature-based methods well known in the art. Some of the potentialspecific methods are described below, but there are other well-knownmethods which will be apparent to those skilled in the art and aredescribed in Fowler, J. and Wolf, A Positron emitter-labeled compoundsin POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M.,Mazziota, J., and Schelbert, H. eds.) p 391-450 (Raven Press, N.Y.)(1986), Coenen, H. et al., Radiochimica Acta 34: 47 (1983), andKulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), thecontents of which are hereby incorporated by reference.

^(99m)Tc derivatives are prepared by complexation with the aryl thiols.Radiolabeling with ¹¹C can be readily done via N-methylation,O-methylation as described above substituting [¹¹C]methyl iodide,[¹¹C]alkylation, or [¹¹C] carboxylation of suitable alkyl, alkenyl, oralkynyl Chrysamine G analogues. [¹⁸F]aryl fluorides derivatives can beprepared by substituting ¹⁸F-labeled precursors such as [¹⁸F]LiB₄ in theSchiemann reaction described above, via triazene decomposition withCs[¹⁸F], or via nucleophilic ¹⁸F-for-X substitution, where X=tosyl,triflate, N₂, ⁺N(CH₃)₃, or halogen. Radiobromination using ⁷⁵Br and ⁷⁶Brcan be accomplished using either electrophilic (Br⁺) or nucleophilic(Br⁻) substitution techniques analogue to radioiodination techniques,see Coenen, H., supra.

Synthesis of the 3-Hydroxy-1,2-benzisoxazole derivative and relatedderivatives (see FIG. 2C)

2,6-Dihydroxybenzoic acid (γ-resorcylic acid) methyl ester (TCI America,Portland, Oreg.) is converted to the hydroxamic acid by the use ofhydroxylamine hydrochloride according to the method of Böshagen (Chem.Ber 100: 954-960; 1967). The hydroxamic acid is converted to thecorresponding 3-hydroxy-1,2-benzisoxazole with the use of SOCl₂ and thentriethylamine, also by the method of Böshagen (Chem. Ber 100: 954-960;1967). This compound is then converted to the formyl derivative andcoupled to the appropriate tetraethyl diphosphonate (which in some casesmay be brominated) as described above under the synthesis of alkenylderivatives. The bromo derivatives can then converted to the tri-alkyltin and iodo-derivatives as described above.

Alternatively, 5-formyl salicylic acid is coupled to the appropriatetetraethyl diphosphonate as usual and the resulting4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylethenyl)-benzene is convertedfirst to the dimethyl ester and then to the hydroxamic acid and finallythe benzisoxazole by the method of Böshagen as described above. A thirdtype of 3-hydroxy-1,2-benzisoxazole is synthesized from several isomericdihydroxy benzenedicarboxylic acids including4,6-dihydroxy-1,3-benzenedicarboxylic acid, 3,6-dihydroxyphthalic acid,and 2,5-dihydroxyterephthalic acid (Aldrich Chem. Co., Milwaukee, Wis.).After formylation and coupling to the appropriate tetraethyldiphosphonate by standard procedures, followed by conversion to thedimethyl esters, the dihydroxy/diesters are converted todihydroxy/dihydroxamic acids by reaction with hydroxylamine by themethod of Böshagen described above. Conversion to the doublebenzisoxazole is effected by treatment with SOCl₂ and triethylamine,again, by the method of Böshagen described above.

Synthesis of the phthalimide or isoindole-1,3(2H)-dione derivative (seeFIG. 2D)

3-Hydroxyphthalimide made from 3-hydroxyphthalic anhydride (AldrichChemical Company, Milwaukee, Wis.) is converted to the formyl derivativeand coupled to the appropriate tetraethyl diphosphonate (which in somecases may be brominated) as described above under the synthesis ofalkenyl derivatives. The bromo derivatives can then converted to thetri-alkyl tin and iodo-derivatives as described above.

Synthesis of the phthalhydrazide or 2,3-benzodiazine-1,4(2H,3H)-dionederivative (see FIG. 2E)

3-Hydroxyphthalhydrazide made from the reaction of 3-hydroxyphthalicanhydride (Aldrich Chemical Company, Milwaukee, Wis.) with hydrazine isconverted to the formyl derivative and coupled to the appropriatetetraethyl diphosphonate (which in some cases may be brominated) asdescribed above under the synthesis of alkenyl derivatives. The bromoderivatives can then converted to the tri-alkyl tin and iodo-derivativesas described above.

Synthesis of the 2,3-benzoxazine-1,4(3H)-dione derivative (see FIG. 2F)

3-Hydroxyphthalic anhydride (Aldrich Chemical Company, Milwaukee, Wis.)is converted to the 2,3-benzoxazine with the use of hydroxylamine. Thebenzoxazine derivative is then converted to the formyl derivative andcoupled to the appropriate tetraethyl diphosphonate (which in some casesmay be brominated) as described above under the synthesis of alkenylderivatives. The bromo derivatives can then converted to the tri-alkyltin and iodo-derivatives as described above.

Synthesis of the (2H)1,3-benzoxazine-2,4(3H)-dione derivative (see FIG.2G)

This compound is synthesized by the method of Effenberger et al., (Chem.Ber. 105: 1926-1942; 1972). Briefly, 4-hydroxybenzaldehyde (Aldrich) iscoupled with the appropriate tetraethyl diphosphonate via the sameprocedure used for formylsalicylic acid derivatives. This adduct is thenconverted to the carbamate by reaction with ethoxycarbonylisocyanate(O═C═N—CO—O—Et) in the presence of triethylamine. This substitutedcarbamate (or N-ethoxycarbonyl-carbamic acid-phenyl ester) is convertedto the benzoxazinedione by heating in diphenyl ether. Thebenzoxazinedione is then converted to the tri-alkyl tin andiodo-derivatives, as described above.

Synthesis of the (3H)2-benzazine-1,3(2H)-dione derivative (see FIG. 2H)

3-Hydroxyphenylacetic acid (Aldrich Chemical Company, Milwaukee, Wis.)is formylated, converted to the amide and then coupled with theappropriate tetraethyl diphosphonate via the same procedure used forformylsalicylic acid derivatives. This adduct is then converted to theN-(3-hydroxyphenylacetoxy)-carbamic acid ethyl ester derivative byreaction with ethyl chloroformate. This substituted carbamate isconverted to the benzazinedione by heating in diphenyl ether. Thebenzazinedione is then converted to the tri-alkyl tin andiodo-derivatives, as described above.

Synthesis of the 1,8-Naphthalimide derivative (See FIG. 2I)

4-Amino-1,8-naphthalimide is converted to the formyl derivative andcoupled to the appropriate tetraethyl diphosphonate (which in some casesmay be brominated) as described above under the synthesis of alkenylderivatives. The bromo derivatives can then converted to the tri-alkyltin and iodo-derivatives as described above.

Synthesis of Tetrazole and Oxadiazole Derivatives (See FIGS. 2J and 2K)

2-Cyanophenol (Aldrich Chemical Company, Milwaukee, Wis.) is convertedto the tetrazole by reaction with sodium or aluminum azide according tothe method of Holland and Pereira J. Med. Chem. 10: 149 (1967) andHolland U.S. Pat. No. 3,448,107. Briefly, 2-cyanophenol or alkenylcyanophenol derivatives of Chrysamine G (made by coupling4-formyl-2-cyanophenol with the appropriate tetraethyl diphosphonate) (1mmol) in 40 ml DMF is treated with sodium azide (10 mmol) andtriethylamine hydrochloride (10 mmol) under argon. The mixture isstirred at 120° C. for 2 hrs after which the mixture is cooled andworked up in a manner analogous to that described above for ChrysamineG.

The oxadiazoles are synthesized by treatment of the tetrazoles preparedas above with an acid anhydride (such as acetic anhydride). An alternatemethod is that of Bamford et al., J. Med. Chem. 38: 3502 (1995). In thisprocedure, hydrazide alkenyl derivatives of Chrysamine G or salicylicacid (obtained by treatment of the respective esters with hydrazine) aretreated with methyl isothiocyanate in the presence ofdicyclohexylcarbodiimide.

Synthesis of Chrysamine G Derivatives for Use as Controls

The aniline derivative is synthesized by substituting two equivalents ofaniline (Fisher Chemical Co., Fair Lawn, N.J.) for each equivalent ofbenzidine. The 2,2′-disulfonic acid derivative is synthesized bysubstituting benzidine-2,2′-disulfonic acid (Pfaltz & Bauer, Inc.,Waterbury, Conn.) for benzidine. The phenol derivative is synthesized bysubstituting one equivalent of phenol for each equivalent of salicylicacid. Congo red (Aldrich certified grade) is obtained commercially.

EXAMPLE 2

Chrysamine G and Chrysamine G Derivatives Bind Specifically to Aβ

Binding to synthetic Aβ(10-43)

Chrysamine G binds well to synthetic Aβ(10-43) peptide in vitro. FIG. 4Ashows a Scatchard analysis of the binding of Chrysamine G to Aβ(10-43).The higher affinity component has a K_(D) of 0.257 μM and a B_(max) of3.18 nmoles Chrysamine G/mg Aβ(10-43). The lower affinity component isless well defined by these data, but appears to have a K_(D) of 4.01 μMand a B_(max) of 18.7 nmoles Chrysamine G/mg Aβ(10-43). The low affinitycomponent represents the binding of Chrysamine G at high concentrationsto a distinct, low-affinity site, not the binding to an impurity in thepreparation. The amount of Chrysamine G injected in vivo is so low thatthere is not any binding to the low-affinity component. At very lowconcentrations, the ratio of high-to-low affinity binding is very large.

The amount of Chrysamine G binding is linear with peptide concentrationover the range employed, as shown in FIG. 5.

Kinetics of Binding

Kinetic studies showed a fairly rapid association (FIG. 6A), essentiallycomplete by 1 min, at a [Chrysamine G]=112 μM with a t_(½) of 8.9±1.8sec and a somewhat less rapid dissociation (FIG. 6C), t_(½)=55±9.4 sec[dissociation rate constant (k⁻¹)=1.26×10⁻² sec⁻¹]. FIG. 6B shows atransformation of the association kinetic data according to the methodof Bennett and Yamamura. Bennett, J. P. and Yamamura, H. I. inNEUROTRANSMITTER RECEPTOR BINDING (N.Y.: Raven Press 1985) pp. 61-89.The linear portion of the association curve in FIG. 6A is transformedinto the line of FIG. 6B, in which ln[B_(eq)/(B_(eq)−B_(t))]is plottedversus time, where B_(eq) is the amount of Chrysamine G bound atequilibrium (4 min) and B_(t) is the amount bound at time=t. The slopeof this line equals k_(observed) and k₁=(k_(observed)−k⁻¹)/[ChrysamineG], where k⁻¹ is the dissociation rate constant determined from the datain FIG. 6C. The curve in FIG. 6C follows the equation:

A _(t) =A ₀e^(−k) ^(⁻¹) ^(t)

where A_(t) is the amount of Chrysamine G remaining bound at time=t, A₀is the amount of Chrysamine G bound at time=0, t is the time in min, andk⁻¹ is the dissociation rate constant. From this analysis, theassociation rate constant (k₁) is calculated to be 3.75×10⁴ M⁻¹ sec⁻¹giving a K_(D)=k⁻¹/k₁=0.34 μM, in good agreement with the Scatchardanalysis.

Chrysamine G Derivatives Can Inhibit the Binding of Chrysamine G to Aβ

K_(i) values for the inhibition of [¹⁴C] Chrysamine G binding toAβ(10-43) by the Chrysamine G analogues are shown under the chemicalstructures in FIG. 1 and several displacement curves are shown in FIG.3. K_(i) is defined as IC₅₀/(1+[L]/K_(D)), where [L] is theconcentration of [¹⁴C] Chrysamine G in the assay (0.100-0.125 μM) andK_(D) is 0.26 μM, the K_(D) of Chrysamine G determined by the Scatchardanalysis above. Chrysamine G itself gives a K_(i) of 0.37±0.04 μM, avalue very consistent with those obtained from the Scatchard and kineticanalyses. Congo red gives a K_(i) of 2.82±0.84 μM. The difluoroderivative of Chrysamine G, (5-FSA)CG, (FIG. 1) is one-third as potentas Chrysamine G itself (K_(i)=1.16±0.19 μM). The activity of thedifluoro Chrysamine G derivative suggests that an ¹⁸F difluoroChrysamine G derivative works for PET imaging and an ¹⁹F difluoroChrysamine G derivative works for MRS/MRI imaging of brain.

The 3-ICG is slightly more potent than Chrysamine G. The activity of the3-ICG derivative suggests that an ¹²³I difluoro Chrysamine G derivativeworks for SPECT imaging. Methylating the phenol of 3-ICG decreases theaffinity by a factor of 10 in 3-IGC(OMe)₂. Methylating the carboxylategroup effected an even greater (about 200-fold) decrease in affinity inCG(COOMe)₂. Removing the acid moiety entirely, as in the phenolderivative, completely destroyed binding affinity.

These results suggest that the acid moiety of Chrysamine G analoguesplays the major role in binding to Aβ and that the phenol moiety playsan facilitating role. The effect of the phenol could occur throughhydrogen bonding to the acid which could serve to stabilize thestructural orientation of the acid moiety. The presence of a phenol inthe ortho position could also alter the charge distribution of the acideither through hydrogen bonding or through changes in the chargedistribution of the aromatic system as a whole. Alternatively, thephenol could directly participate in binding to the amyloid via abi-dentate attachment of both the phenol and the acid to the amyloidbinding site. Adding a second phenol ortho to the carboxylate as in theresorcylic acid derivative, (6-OHSA)CG, produces the highest affinitycompound in this series having a K_(i) of 0.094±0.02 μM.

Increasing the lipophilicity of the biphenyl backbone appears toincrease the affinity somewhat. The di-halo derivatives, 3,3′-I₂CG,3,3′-Br₂CG, and 3,3′-Cl₂CG, all have very similar K_(i) values which areabout half that of Chrysamine G.

Distorting the dihedral angle between the phenyl rings of the biphenylgroup by substitution at the 2-position markedly diminishes affinity.This is demonstrated by the inactivity of the 2,2′-di-sulfonic acidderivative of Chrysamine G, 2,2′-(SO₃)₂CG. Since the 3,3′di-carboxylicderivative, 3,3′-(COOH)₂CG, shows only a 7-fold loss of activity fromChrysamine G, it is unlikely that the additional acidic moieties are thesole cause for the loss of activity in the 2,2′-disulfonic acid. This2,2′- derivative is unique in that the bulky sulfonate groups in the2-position force the biphenyl group out of planarity. Molecularmodelling studies showed that the dihedral angle between the twobiphenyl benzene rings in the 2,2′-disulfonic acid derivative is 83°.This angle is approximately 35-40° in Chrysamine G and all of the otheractive derivatives.

In an attempt to explore the importance of the bidentate nature of thefunctional groups of Chrysamine G, the binding of an aniline derivativewhich represents one-half of a Chrysamine G molecule (FIG. 1) wasstudied. An approximation of the energy of binding can be calculatedfrom the equation:

ΔG=−RT 1n K _(eq)

where ΔG is the energy of the binding reaction, R is the molar gasconstant [8.31441 J/(mole•° K)], T is temperature in ° K and K_(eq) isthe equilibrium constant for the reaction:

[probe]+[peptide]⇄[probe•peptide]

and K_(eq)=1/K_(D)⇄1/K_(i). Using the value of 0.26 μM for the K_(D) ofChrysamine G, the energy of binding is roughly 38 KJ/mole. If theaniline derivative binds with one-half of this energy, the expectedenergy of binding would be about 19 KJ/mole. From the K_(i) of 73 μM forthe aniline derivative, the energy of binding is 23 KJ/mole which is inacceptable agreement with the predicted value. The importance of thehydrophobic region of Chrysamine G and the aniline derivative isdemonstrated by the total lack of binding activity of salicylic aciditself.

The affinity of Chrysamine G for Aβ appears to be several fold greaterthan the affinity of Congo red for this peptide. The binding isreversible with a dissociation constant of approximately 250-400 nM,whether measured by Scatchard analysis, kinetic methods, or inhibitionof binding. Owing to the non-crystalline, poorly soluble nature ofamyloid fibrils, the structure of Congo red or Chrysamine G complexeswith amyloid has never been defined by precise structural techniquessuch as x-ray crystallography or multi-dimensional NMR. Models of Congored interactions with amyloid have been proposed. Cooper, Lab. Invest.31: 232 (1974); Romhanyi, Virchows Arch. 354: 209 (1971). This worksuggests that Congo red does not bind to a single amyloid peptidemolecule, but binds across several Aβ molecules oriented by virtue ofthe beta-sheet fibril. Klunk et al., J. Histochem. Cytochem. 37: 1273(1989).

FIG. 7 shows a schematic of this model, generated using MacroModel 2.5,in which Chrysamine G spans 5 peptide chains in an anti-parallelbeta-sheet conformation. The peptides are used without furtherstructural refinement. The peptides are aligned so that alternate chainswere spaced 4.76 Å apart, characteristic of beta-sheet fibrils.Alternate peptide chains are drawn in black and white. Chrysamine G(black) is energy minimized and aligned with the fibril model tomaximize contact with lysine-16 (light grey ovals in top figure) and thehydrophobic phenylalanine 19/20 region (bottom). The two views are ofthe same model at approximately 90° from one another. The white arrowsindicate the direction taken to obtain the alternate view.

The 19.1 Å spacing between the carboxylic acid moieties of Chrysamine Gmatches well with the distance of 19.0 Å across the 5 chains (4×4.76 Åbetween adjacent chains shown by Kirschner et al., Proc. Natl. Acad.Sci. U.S.A. 83: 503 (1986)). If the native structure of Aβ involves ahairpin loop structure as Hilbich et al., suggest (Hilbich et al., J.Mol. Biol. 218: 149 (1991)), then chains 1 and 2, 3 and 4, 5 and 6,etc., would be folded halves of the same molecule, but the model wouldotherwise be the same. Also important to note is the necessity forpositively charged amino acid residues in this model, such as lysine-16in Aβ. Previous work has shown that Congo red binding correlates wellwith the number of positively charged amino acids in a sample of amyloidfibrils. Klunk et al., J. Histochem. Cytochem. 37: 1273 (1989). Thebidentate nature of the model in FIG. 7 and the importance ofhydrophobic interactions is supported by the decrease in affinity of themono-dentate aniline analogue of Chrysamine G and the inactivity ofsalicylic acid as well as the increased potency of the more lipophiliccompounds having two halogens on the benzidine moiety (see FIG. 1). Theimportance of the nearly planar biphenyl group is suggested by theinactivity of the 2,2′-disulfonic acid derivative.

EXAMPLE 3

Chrysamine G Distinguishes Alzheimers's Disease Brain from Normal Brain

Characterization of the Binding of Chrysamine G to AD Brain

Scatchard analyses of the binding of Chrysamine G and Chrysamine Gderivatives to AD brain samples were performed in an effort tounderstand the increased binding of Chrysamine G to AD brain. (FIG. 4B,Table 1). Under the conditions employed, control and AD brain showed asingle binding component. The K_(D) in AD brain was 16% lower thancontrol but the difference was not significant (p=0.29). The B_(max) inAD brain was 36% higher than the B_(max) in control brain, but, again,the difference did not reach significance (p=0.09). Therefore, theincreased binding in AD brain appears to be mainly due to the presenceof more of the same binding component which exists in control brain,rather than the presence of a unique component.

TABLE 1 Comparison of binding Parameters in AD and control brain B_(max)(pmol/μg K_(D) (μM) prot) Control 0.47 ± .049 0.576 ± .092 (n = 6) AD(n= 5) 0.39 ± .048 0.784 ± .061

The binding of CG to AD brain significantly correlated with numbers ofNPs in the association cortices of the brain. FIG. 8A shows thecorrelation of [¹⁴C]CG binding with numbers of NPs in thesuperior/middle frontal and superior temporal cortex of AD brain. Thecorrelation with NPs was significant whether controls were included(r=0.69; p=0.001) or if the AD brains were considered alone (r=0.59;p=0.007). FIG. 8B shows a similar correlation with NFT counts. As withNPs, the correlation with NFTs was significant whether controls wereincluded (r=0.60; p=0.001) or if the AD brains were considered alone(r=0.50; p=0.026). The correlation with NFTs is not surprising since CGis a derivative of Congo red, which stains NFTs. The number of NPs wassignificantly correlated with the number of NFTs (r=0.82; p=0.0001).

Only qualitative data on the presence or absence of amyloid angiopathywas available for the brains used in this study, so similar correlationscould not be performed between CG binding and cerebrovascular amyloidlevels. The presence of amyloid angiopathy does appear to be aconfounding variable in the correlation of CG binding with NP counts.FIG. 8A shows the improved correlation of CG binding to NP counts inbrains without amyloid angiopathy (r=0.79; p=0.01) compared to thosebrains with cerebrovascular amyloid deposits (r=0.49; p=0.15). A similarimprovement was not found in the correlation to NFT counts.

The K_(D) for [¹⁴C] Chrysamine G binding to AD brain is similar to thatfound for [¹⁴C] Chrysamine G binding to synthetic Aβ in vitro,suggesting that binding in brain homogenates also may representinteraction with Aβ. The correlation of Chrysamine G binding to NFTs mayindicate that Chrysamine G binds to these structures in brainhomogenates as well. Alternatively, since the number of NFTs correlatesclosely with the number of NPs, the correlation of [¹⁴C] Chrysamine Gbinding to NFTs may just be an epiphenomenon of Chrysamine G binding toNPs.

The useful Chrysamine G derivatives or analogues provided herein havebinding affinities that are at least in the range of 0.01 to 10.0 μMK_(D), as measured by binding to either synthetic Aβ peptide orAlzheimer's Disease brain tissue; higher affinity compounds havingbinding affinities in the range of 0.0001 to 0.01 μM are also useful inthe method of the present invention.

Considering the above, Chrysamine G binding may not be specific for Aβ.Instead, Chrysamine G binding may reflect the total amyloid “load” ofthe brain, comprised of aggregated deposits of Aβ in neuritic plaquesand cerebrovascular amyloid. Deposits of phosphorylated tau protein inNFTs may contribute to Chrysamine G binding as well. Goedert, M. et al.,PNAS 85: 4051 (1988). NFTs also are composed of anti-parallel beta-sheetfibrils similar in quaternary structure to fibrils of Aβ. Kirschner etal., Proc. Natl. Acad. Sci. U.S.A. 83: 503 (1986).

Total and Relative Chrysamine G Binding Distinguishes AD From NormalBrain

In vitro binding assays such as those described above and below arewidely used in the art as models to screen compounds for in vivo bindingin brain and to predict success in subsequent in vivo imaging studies.See, Young, A. et al., Receptor Assays: In Vitro and In Vivo. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) pp. 73-111 (1986). The labeled Chrysamine Gand Chrysamine G derivatives of the invention also may be used in the invitro binding assays described above and below to quantitate amyloiddeposits in biopsy or post-mortem specimens.

Saturable (specific) binding of [¹⁴C] Chrysamine G was observed both inAD brain and control brain homogenates and constituted 60-80% of totalbinding in AD brain. Non-saturable binding was very similar in AD andcontrol brain. Both saturable and total binding were greater in AD brainthan in control. Despite the lower sensitivity obtained when using totalbinding, this parameter is more predictive of success in in vivo studieswhich are the ultimate goal of this invention. Also for the purpose ofextension to in vivo studies, it is advantageous if Chrysamine G bindingin cortical areas is normalized to a brain area in which Chrysamine Gbinding is very similar in both AD and control brain. This obviates theneed to calculate the absolute quantity bound which is difficult to doin vivo. We examined binding in the cerebellum as a potential controlarea because classical NPs are exceedingly rare in this brain area(Joachim et al., Am. J. Pathol. 135: 309 (1989)).

The average amount of [¹⁴C] Chrysamine G bound to control cerebellum isnearly identical to the amount bound to AD cerebellum (Table 2),supporting the use of cerebellum as an internal control. Therefore, thecerebellar ratio (CBR) accurately reflects the absolute quantity of[14C] Chrysamine G bound and offers the advantage of providing aninternal control for each brain. Binding is greater in AD brain whetherexpressed in absolute terms of fmol/μg protein (Table 2) or as a ratioto the binding in the cerebellum of the same brain (Table 3). The CBR isthe more sensitive measure and shows less variability between brains.The use of total binding and CBRs greatly facilitates extension of theseex vivo results to in vivo studies. Accordingly, the results below areexpressed in these terms whenever appropriate.

TABLE 2 Comparison of total binding in AD and control brain* Control ADBrain Area (fmol/μg protein) (fmol/μg protein) p Value Cerebellum 75 ±13 (n = 8)  73 ± 9 (n = 11) p = 0.91 Frontal Pole 58 ± 8 (n = 6) 124 ±16 (n = 10) p < 0.006 Superior/Middle 54 ± 10 (n = 8) 130 ± 21 (n = 11)p < 0.005 Frontal Superior 66 ± 17 (n = 8) 121 ± 14 (n = 11) p < 0.02Temporal Head of Caudate 73 ± 11 (n = 4) 123 ± 22 (n = 7) p = 0.14Inferior 76 ± 13 (n = 8) 137 ± 19 (n = 11) p < 0.03 Parietal Occipital64 ± 16 (n = 8)  95 ± 12 (n = 11) p = 0.1 *High and low plaque AD brainscombined.

TABLE 3 Comparison of total binding in AD and control brain as a ratioto cerebellum* Control AD Brain Area (CBR) (CBR) p Value Frontal Pole0.87 ± .04 (n = 6) 1.87 ± .25 (n = 10) p < 0.004 Superior/Middle 0.73 ±.02 (n = 8) 1.84 ± .18 (n = 11) p < 0.001 Frontal Superior 0.86 ± .08 (n= 8) 1.63 ± .17 (n = 11) p < 0.002 Temporal Head of Caudate 0.95 ± .04(n = 4) 1.76 ± .31 (n = 7) p < 0.04 Inferior 0.90 ± .08 (n = 8) 1.93 ±.20 (n = 11) p < 0.003 Parietal Occipital 0.77 ± .13 (n = 8) 1.44 ± .20(n = 11) p < 0.02 *The CBR for each sample is obtained by dividing theabsolute value of [¹⁴C] Chrysamine G binding in that sample by theabsolute value of [¹⁴C] Chrysamine G binding in the cerebellar samplefrom that same brain. The values in the table are the average CBRs fromeach brain area (± SEM). High- and low-plaque AD brains combined.

FIGS. 9A and 9B shows the binding of [¹⁴C] Chrysamine G to six brainareas normalized to the cerebellum of the same brain. The binding ofChrysamine G to AD brain areas in AD brains having more than 20 NPs/×200magnification, “High Plaque AD Brains”, is shown in FIG. 9A. The bindingof Chrysamine G to AD brain areas in AD brains having less than 20NPs/×200 magnification, “Low Plaque AD Brains”, is shown in FIG. 9B. Inall brain areas, the binding to AD brain is significantly greater thanthe binding to control (see Table 3). In superior/middle frontal cortex,there is no overlap between control and any of the AD samples. In allbrain areas except the occipital cortex, there is no overlap betweencontrol and the AD samples having >20 NPs/×200 magnification. In brainareas with the least deposition of classical NPs, such as the occipitalcortex (and cerebellum), the greatest overlap between AD and control wasobserved.

FIG. 9C shows the data from two patients who had Down's syndrome. Down'ssyndrome patients all develop deposits of Aβ by their fourth decade andmany develop AD. Wisniewski et al., Neurology 35: 957 (1985); Schapiroet al., Neurobiol. Aging 13, 723 (1992). Both of these patients showed[¹⁴C] Chrysamine G binding above the control range. Since the youngerpatient (23 years old) had amyloid deposits but was not yet clinicallydemented, FIG. 9C suggests that Chrysamine G can detect differences fromcontrol in non-demented patients destined to develop AD long before thedementia is clinically evident.

The compounds and method of the invention provide two usefulmeasurements for differentiating AD brain from normal brain; either (1)total Chrysamine G binding (Table 2) or (2) the ratio of Chrysamine Gbinding in a given brain area to binding in the cerebellum of the samebrain (Table 3). These measurements furnish two great advantages for invivo quantitation of AD neuritic plaques. First, by providing a means tomeasure total Aβ binding, rather than specific Aβ binding, the instantinvention can quantify Aβ deposition without having to expose thesubject to a second injection of radioactive material in order tomeasure non-specific binding. Because of this, the data are expressed astotal binding only. In all of the experiments presented, specificbinding data yields even greater differences between AD and controlbrain.

Second, variations in brain uptake of Chrysamine G derivatives willaffect the absolute concentration of Chrysamine G in brain. Somemechanism will be necessary, therefore, to account for these variationsbetween subjects. Each patient can serve as his/her own control byfinding a brain area that shows little Aβ deposition (i.e., anexperimental “blank”). Since classical NPs are exceedingly rare in thecerebellum (Joachim et al., Am. J. Pathol. 135: 309 (1989)), ChrysamineG binding to the cerebellum was used as a control for each brainstudied. The results were expressed in terms of the ratio of ChrysamineG binding in a given brain area to binding in the cerebellum of the samebrain (FIG. 9 and Table 3).

For the purposes of in vivo quantitation of amyloid in AD, the effect ofbrain atrophy should be considered. Therefore, when using the ChrysamineG and Chrysamine G derivative probes in vivo to quantitate amyloid,brain atrophy can be corrected based on MRI volume measurements. MRIvolume measurements performed in conjunction with the method of theinvention are analogous to those routinely employed in the art. See,Pearlson, G. and Marsh, L. MAGNETIC RESONANCE IMAGING IN PSYCHIATRY inAnnual Review of Psychiatry (Vol. 12) Oldham, J. et al., eds. p. 347-381(1993). Therefore a method for determining the total radioactivity pervolume of brain area would use the following equation:$\frac{{total}\quad {SPECT}\quad {or}\quad {PET}\quad {signal}\quad {from}\quad {brain}\quad {area}\quad {``A"}}{\begin{matrix}{{MRI}\quad {determined}\quad {brain}\quad {volume}} \\{\left( {{excluding}\quad {CSF}} \right)\quad {in}\quad {brain}\quad {area}\quad {``A"}}\end{matrix}}$

Designating this measurement as the signal/volume for brain area “A” orS/V_(A) means that the cerebellar ratio would be expressed as:${Ratio}_{A} = \frac{S/V_{A}}{S/V_{CB}}$

where S/V_(CB) is the signal/volume in the cerebellum of the samesubject during the same imaging study. This ratio from any brain areaother than cerebellum from a patient suspected of having AD or otherpathological condition characterized by the deposition of amyloid couldthen be compared to the normal range of the analogous ratio from thesame brain area of a group of age-matched normal control subjects. Theratio of the binding to brain areas with high deposits of neuriticplaques to the cerebellum can be used as the parameter to distinguishAlzheimer from control subjects.

EXAMPLE 4

The Octanol-Water Partition Coefficients of Chrysamine G, Chrysamine GDeriviatives, and Congo Red

The octanol-water partition coefficient is a measure of the relativelipophilicity of a compound.

The more lipophilic a compound, the more likely it is to cross theblood-brain barrier. See, Goodman and is Gilman's THE PHARMACOLOGICALBASIS FOR THERAPEUTICS (7th Ed.). The octanol/water partitioncoefficient of Chrysamine G is 60.22±3.97 and that of Congo red is0.665±0.037 (p<0.001). This suggests that Chrysamine G is approximately90 times more lipophilic than Congo red and therefore is theoreticallymore likely to cross the mammalian blood-brain barrier. Theoctanol/water partition coefficients for the 3-iodo and 3,3′-diiododerivatives of Chrysamine G (FIG. 1) are 72.53±0.74 and 112.9±7.3,respectively. These octanol/water partition coefficients show that thesederivatives, which are non-radioactive analogues of some of theradiolabeled Chrysamine G derivatives to be used for in vivo studies,are up to 170 times more lipophilic than Congo red and up to twice aslipophilic as Chrysamine G. This suggests they will enter the brain muchbetter than either Congo red or Chrysamine G.

EXAMPLE 5

The Ability of Chrysamine G and Chrysamine G Derivatives to Cross theBlood-Brain Barrier and Metabolism of Chrysamine G

Use of the amyloid probes to diagnose AD in vivo requires them to beable to cross the blood-brain barrier and gain access to parenchymalamyloid deposits.

The ability of Chrysamine G to cross the blood-brain barrier was studiedin Swiss-Webster mice. After i.v. injection, the brain/blood ratiomeasured at 15 min was over 10:1 and approached 20:1 by 35 min (FIG.10). The radioactivity in brain stayed nearly constant over this period,but decreased in the blood and increased in the liver. The brain/kidneyratio was highest at 15 min (over the time points sampled) andapproached 0.5. When brain and liver were extracted 60 min after i.v.injection of [¹⁴C] Chrysamine G, >95% of the recovered radioactivityco-eluted with authentic Chrysamine G on reverse phase HPLC, indicatingno significant metabolism of Chrysamine G over this period of time.

Chrysamine G does get into normal mouse brain, and the brain/blood ratiois high. The radioactivity in brain remained relatively constant overthe first 30 min while decreasing in blood and increasing in liver. Thissuggests that the high brain/blood ratio is more a result of efficientremoval of Chrysamine G from the blood by the liver than to furtheraccumulation in the brain. At 60 min, essentially all of theradioactivity found in the brain and liver proved to be unchangedChrysamine G. Congo red does not cross the blood-brain barrier well.Tubis et al., J. Amer. Pharm. Assn. 49: 422 (1960). Most of the Congored is cleared by the liver and spleen and the brain/kidney ratioachieved in guinea pigs is approximately 0.07. Tubis et al., supra.Chrysamine G also is cleared by the liver, but has greater entry intothe brain.

In vivo animal testing provides yet a further basis for determiningdosage ranges, efficacy of transfer through the blood barrier andbinding ability. Particularly preferred for this purpose are thetransgenic mouse model of Games et al., (Nature 373: 523 (1995)) orHsiao et al. Science 274: 99-102 (1996) and the “senile animal” modelfor cerebral amyloidosis; i.e., animals such as the transgenic mice oraged dogs or monkeys, which are known to develop variable numbers ofAlzheimer-type cerebral neuritic plaques, see Wisniewski et al., J.Neuropathol. & Exp. Neurol. 32: 566 (1973), Selkoe et al., Science 235:873 (1987), are tested for binding and detection efficacy. This in vivoassay requires control-biopsy or necropsy monitoring to confirm andquantify the presence of amyloid deposits.

Other suitable animal models for use in testing the compositions andmethods of the present invention are produced transgenically. Forinstance, Quon et al., Nature, 352: 239-241 (1991) used ratneural-specific enolase promoter inhibitor domain to prepare transgenicmice. See also, Wirak et al., Science, 253: 323-325 (1991). Still othermodels have been produced by Intracranial administration of the β/A4peptide directly to animals (Tate et al., Bull. Clin. Neurosci., 56:131-139 (1991).

It is noted that none of the in vivo animal models may turn out to beextremely good models for AD neuropathology. Instead, they may moreclosely model the amyloid deposition of normal aging. This isparticularly true of the aged-mammal models. All of these models show apreponderance of diffuse plaques as discussed above for the aged dogmodel. While there is some cerebrovascular amyloid, there are fewneuritic plaques, except in the Games et al. and Hsiao et al. transgenicmouse models. The other transgenic mouse models often show only diffuseplaques. Therefore, while these models may be useful for studyingdistribution of the probes in the brain, there is a fairly lowprobability that these models would show the same quantitativedifferences that would be expected to be seen in AD brain based on thein vitro studies of Chrysamine G binding to AD brain described above.

Evaluating the Ability of Alkyl, Alkenyl or Alkynyl Chrysamine GDerivatives to Cross the Human Blood-Brain Barrier

A dose of approximately 10 mCi of an appropriately radiolabeledderivative of Chrysamine G with a specific activity of approximately 500Ci/mole or higher is injected intravenously into normal subjects andpatients suspected of having AD and monitored by SPECT or PET imaging toanalyze the detectability of the derivative in brain relative to otherorgans and to define the time course of detectability in the brain. Adose which can be reliably detected is defined as a “imaging effectivedose.”

Evaluation of Alkyl, Alkenyl or Alkynyl Chrysamine G Derivatives toDistinguish AD from Age-Matched Controls in Humans

An imaging-effective dose of an appropriately, radioactively labeledderivative of Chrysamine G is injected into a subject suspected ofhaving brain amyloid deposition due to pathological conditions such asAD. After a period of 15 minutes to 24 hours, the radioactive signalfrom brain is detected by SPECT or PET. Radioactivity is simultaneouslydetected in all brain areas included in the field of view of thedetector. This field of view will be set up so as to include largeportions of the cerebellum, superior temporal cortex, superior/middlefrontal cortex, and intervening brain regions. An MRI scan will beperformed prior to the study so that corrections can be made for brainatrophy in the areas of interest by methods discussed in Example 3. TheS/V_(A), S/V_(B), and Ratio_(A) variables discussed in Example 3 will becalculated and compared to analogous normative ratios obtainedpreviously from age-matched normal control subjects.

EXAMPLE 6

Historical Localization of the Binding of an Alkenyl Chrysamine GDerivative to Amyloid

The top frame of FIG. 11 demonstrates a human AD brain stained by1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene. The staining methodwas that of Stokes and Trickey, J. Clin. Pathol. 26: 241-242 (1973) with1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene substituted for Congored. This staining is much more intense than that observed withChrysamine G, Congo red, or Thioflavin S. Numerous amyloid plaques andneuropil threads can be readily identified as well as a neurofibrillarytangle. The bottom photomicrograph in FIG. 11 shows a section oftrangenic mouse brain [Tg(HuAPP695.SWE)2576; Hsiao et al. Science 274:99-102 (1996)] stained with 1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene. The dense amyloid plaques isintensely stained. Cerebrovascular amyloid also is intensely stained inboth human and mouse brain (data not shown).

EXAMPLE 7

Assessing the Toxicity of Alkyl, Alkenyl or Alkynyl Chrysamine GDerivatives

At doses of 10 and 100 mg/kg non-radioactive Chrysamine G administeredintraperitoneally, no notable behavioral effects or toxicity wereobserved in mice for periods up to 72 hrs. The doses of [¹⁴C] ChrysamineG administered were on the order of 1 mg/kg.

Chrysamine G appeared to show little acute toxicity based on attempts toestablish an LD₅₀. Even when the maximum volume that can be injectedinto a mouse without harming it just from fluid volume effects (approx.0.025 ml/g) of a saturated solution of Chrysamine G was injected intomice (100 mg/kg), there were no behavioral changes noted for at least 72hrs, the longest period tested. Doses required for detection ofradiolabeled derivatives by SPECT or PET would be orders of magnitudebelow this dose.

Congo red has been safely injected into humans in quantities muchgreater than would be used for the radioactive Chrysamine G derivatives.The LD₅₀ for Congo red has been shown to be 190 mg/kg mouse (Tubis etal., J. Amer. Pharm. Assoc. 49: 422 (1960)), which is similar tothe >100 mg/kg LD₅₀ shown for Chrysamine G. Thus, these two chemicallysimilar compounds cause similar low toxicities in mice.

Other alkyl, alkenyl or alkynyl Chrysamine G derivatives can similarlybe tested for toxicity in mice and other higher mammals by injecting awide range of concentrations and studying the animals for various signsof toxicity by methods well known in the art. See, Goodman and Gilman'sTHE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

EXAMPLE 8

Assessing the Ability of Chrysamine G to Protect Against Aβ(25-35)-Induced Toxicity

Protection from Aβ (25-35)-induced Toxicity in PC-12 cells

Rat pheochromocytoma cells (PC-12) were grown in RPMI 1640 media with10% fetal bovine serum. Approximately 5,000 exponentially growing cellswere plated in 96-well plates in a volume of 100 μl of media and allowedto incubate at 37° C. overnight. The Aβ (25-35), which had beenpre-aggregated at 37° C. for 7 days was pre-incubated with Chrysamine G(CG) or related compounds in aqueous solution prior to addition of 20 μlto achieve the final concentrations given (0.01 to 10 μM Aβ(25-35) and0.03 to 20 μM CG). The cells were incubated for 24 hrs prior to theaddition of 13.3 μl of 5 mg/ml MTT(3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) in sterilephosphate buffered saline. After 4.5 hrs at 37° C., 100 μl of extractionbuffer (20% w/v SDS in 50% DMF/water; pH adjusted to 4.7 with 2.5% of80% acetic acid and 2.5% 1N HCl) was added and the plates were incubatedovernight. Hansen et al., J. Immunol. Methods 119: 203 (1989). Colordevelopment was then measured at 560 nm. Maximum viability was definedas the absorbance of control wells to which only the 20 μl of distilled,deionized H2O was added. Maximum toxicity was defined by wells in whichthe cells were lysed by addition of 0.1% (final concentration) of TritonX-100.

Incubation of PC12 cells with Aβ(25-35) results in aconcentration-dependent decrease in the ability of these cells to reduceMTT (FIG. 12). FIG. 12 shows the effect of increasing concentrations ofAβ(25-35) in the presence and absence of Chrysamine G on the cellularredox activity of PC12 cells as measured by MTT reduction. The reductionproduct of MTT absorbs at 560 nm which is plotted on the vertical axis.The effect of Aβ(25-35) alone is shown in the filled bars and shows adose dependent decrease in MTT reduction. Significant differences fromcontrol (no Aβ, no Chrysamine G) are shown in white numbers inside thefilled bars. The protective effect of 20 μM Chrysamine G is shown in theopen bars. Significant differences between MTT reduction in the presenceand absence of Chrysamine G are shown in black numbers inside the openbars.

FIG. 13 demonstrates the protective effect of increasing concentrationsof Chrysamine G against the Aβ(25-35)-induced reduction of cellularredox activity of PC12 cells. The effect of Chrysamine G in the absenceof Aβ(25-35) is shown in the filled bars. There was no significantdifference between control (no Aβ, no Chrysamine G) and any of theconcentrations of Chrysamine G in the absence of Aβ(25-35). MTTreduction in the presence of 1 AM Aβ(25-35) and increasingconcentrations of Chrysamine G is shown in the open bars. Significantdifferences in MTT reduction between the presence and absence ofAβ(25-35) at each concentration of Chrysamine G are shown in whitenumbers inside the filled bars. Significant differences in MTT reductionbetween the Aβ(25-35) control (no Chrysamine G) and Aβ(25-35) plusincreasing concentrations of Chrysamine G are shown in black numbersinside the open bars.

As has previously been reported, Congo red protects against theAβ-induced toxicity at concentrations over 2 μM, achieving completeprotection by 20 μM. Burgevin et al. NeuroReport 5: 2429 (1994); Lorenzoand Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994); Pollack et al.,Neuroscience Letters 184: 113 (1995); Pollack et al. NeuroscienceLetters 197: 211 (1995). Chrysamine G shows a protective effect which isdependent on both the concentration of Aβ(25-35) (FIG. 12) as well asthe concentration of Chrysamine G (FIG. 13). The protective effect ofChrysamine G is evident at 0.2 μM, a concentration very close to the Kiof Chrysamine G for binding to synthetic Aβ, 0.37 μM (FIG. 1).Chrysamine G appears to be more potent than Congo red, showing effectsin the range of 0.1 to 1.0 μM. This is consistent with the Ki values forbinding to synthetic Aβ of 0.37 μM for Chrysamine G and 2.8 μM for Congored (FIG. 1).

In another experiment (FIG. 14), the effect of Chrysamine G and thephenol derivative (see FIG. 1), which does not bind Aβ, was examined incells incubated with 1 μM Aβ(25-35). Chrysamine G showed protectiveeffects at 0.1 and 1 μM, but the phenol derivative showed no protectiveeffects, and perhaps enhanced the toxicity of Aβ.

These results suggest that the lipophilic derivative of Congo red,Chrysamine G, prevents Aβ-induced cytotoxicity in cell culture atconcentrations very similar to those at which it binds Aβ. Thisprotection shows structural specificity since the phenol derivativewhich does not bind to synthetic Aβ also does not prevent Aβ-inducedcytotoxicity. Since Chrysamine G partitions into the brain well, theseresults provide evidence that Chrysamine G and Aβ-binding derivatives ofChrysamine G have therapeutic potential in the treatment of AD.

The mechanism of the protective effect of Chrysamine G is unknown atpresent. Two broad possibilities exist. First, Chrysamine G couldinterfere with the aggregation of Aβ. Second, Chrysamine G couldinterfere with the effects (direct or indirect) of Aβ on the targetcells. Congo red does inhibit aggregation of Aβ as well as protectagainst the toxic effects of aggregated Aβ. Lorenzo and Yankner, Proc.Natl. Acad. Sci. 91: 12243 (1994). Interference with aggregation isunlikely in the above experiment since the Aβ was pre-aggregated priorto incubation with Chrysamine G. Thus, inhibition of aggregation couldprove to be an important therapeutic effect of Chrysamine G but is not alikely explanation for the protective effects of Chrysamine G againstpre-aggregated Aβ. The model of Chrysamine G binding to Aβ described inFIG. 7, displays how Chrysamine G could “coat” the surface of Aβ. Thismay change how the fibrillar deposits are recognized by cell-surfacereceptors or other macromolecules such as complement proteins andinterfere with the toxic effects of Aβ which may be mediated by thesemacromolecules. It is likely that Chrysamine G and Congo red exertmultiple effects, both before and after the aggregation of Aβ. This isadvantageous from a therapeutic point of view since patients are likelyto present at a time when there are pre-existing Aβ aggregates as wellas ongoing amyloid deposition.

What is claimed is:
 1. An amyloid binding compound of Formula I or awater soluble, non-toxic salt thereof:

X is C(R″)₂, wherein each R″ independently is H, F, Cl, Br, I, a loweralkyl group, (CH₂)_(n)OR′ wherein n=1, 2, or 3, CF₃, CH₂—CH₂F,O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph), wherein R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R″, a tetrazole oroxadiazole of the form:

wherein R′ is H or a lower alkyl group,  or X is CR′═CR′, N═N, C═O, O,NR′, where R′ represents H or a lower alkyl group, S, or SO₂; each R₁and R₂ independently is selected from the group consisting of H, F, Cl,Br, I, a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃,CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂, tetrazole and oxadiazole of the form:

wherein R′ is H or a lower alykl group, or a triazene of the form:

each Q is independently selected from one or the following structures:IA, IB, IC, ID, IE, IF, and IG, wherein IA has the following structure:

wherein: each of R₃, R₄, R₅, R₆, or R₇ independently is defined the sameas R₁ above, provided that: i) if the compound of Formula I is:

then at least one of R₁-R₄ or R₆-R₇ is independently selected from thegroup consisting of F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′,wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F,O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, tri-alkyl tin, R_(ph) , CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph),wherein R′ is H or a lower alkyl group and wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂ above, tetrazole and oxadiazole of the form:

or a triazene of the form:

ii) if the compound of Formula I is:

wherein R₂ is a lower alkyl group, OR′ (wherein R′ is a lower alkylgroup) or Cl, then at least one other of R₁ -R₇ is independentlyselected from the group consisting of F, Br, I, (CH₂)_(n)OR′, whereinn=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F,(C═O) —R′, N(R′)₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, tri-alkyl tin,CR′═CR′—R_(ph), CR₂, CR₂′—R_(ph), wherein R′ is H or a lower alkylgroup, unless indicated and whrein R_(ph) represents an unsubstitutd orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined from R₁ and R₂ above,tetrazole and oxadiazole of the form:

wherein R′ is H or a lower alkyl group, or a traizene of the form:

iii) if the compound of Formula I is

wherein R₃ or R₇ is a lower alkyl group, CN, F, Cl, or Br, then at leastone other of R₁-R₇ is independendtly selected from the group consistingof F, Br, I, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F,O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, tri-alkyl tin, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R′ is H or a lower alkyl groupand wherein R_(ph) represents an unsubstituted or substituted phenylgroup with the phenyl substituents being chosen from any of thenon-phenyl substituents defined from R₁ and R₂ above, tetrazole andoxadiazole of the form:

wherein R′ is H or a lower alkyl group, or a triazene of the form:

iv) if the compound of Formula I is

wherein R₅ is H, a lower alkyl group, CN, COOR′, (C═O)N(R′)₂, F, Cl, orBr, then at least one other of R₁-R₇ is independently selected from thegroup consisting of I, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃,CH₂—CH₂F, O—CH₂—CH₂F, CH₂CH₂—CH₂F, O—CH₂—CH₂—CH₂F, (C═O)—R′, N(R′)₂,NO₂, O(CO)R′, OR′, SR′, tri-alkyl tin, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph), wherein R′ is H or a lower alkyl group and whereinR_(ph) represents an unsubstituted or substituted phenyl group with thephenyl substituents being chosen from any of the non-pheny substituentsdefined from R₁ and R₂ above, tetrazole and oxadiazole of the form:

wherin R′ is H or a lower alkyl group, or a triazene of the form:

v) if the compound of Formula I is

wherein R₅ is N(R′)₂ (wherein R′ is a lower alkyl group) and R₁, R₂, R₄,and R₆ are all H, then R₃ and R₇ are independently selected from thegroup consisting of F, Cl, Br, I, (CH₂)_(n)OR′, wherein n=1, 2, or 3,CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′ (wherein R′ is H), SR′, COOR′(wherein R′ is H), tri-alkyl tin, R_(ph), CR′═CR′—R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R′ is H or a lower alkylgroup, unless indicated, and wherein R_(ph) represents an unsubstitutedor substituted phenyl group with the phenyl substituents being chosenfrom any of the non-phenyl substituents defined from R₁ and R₂ above,tetrazole and oxadiazole of the form:

or a triazene of the form:

vi) if the compound of Formula I is:

wherein R₅ is H, (CH₂)_(n)OR′ (wherein n is 1 and R′ is H) or COOR′(wherein R′ is a lower alkyl group), then at least one of R₁-R₄, R₆ orR₇ is independently selected from the group consisting of F, Cl, Br, I,a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F,O—CH₂—CH₂F, CH₂—CH₂CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′ (wherein R′ is H), tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R′ is H or a loweralkyl group, unless indicated, and wherein R_(ph) represents anunsubstituted or substituted phenyl group with phenyl substituents beingchosen from any of the non-phenyl substituents defined from R₁ and R₂above, tetrazole and oxadiazole of the form:

or a triazene of the form:

vii) if the compound of Formula I is:

wherein at least one of R₃, R₅ or R₇ is a H, CN, COOR′, or (C═O)N(R′)₂,then at least one other of R₁-R₇ is independently selected from thegroup consisting of F, Cl, Br, I, (CH₂)_(n)OR′, wherein n=1, 2, or 3,CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, (C═O)R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, tri-alkyl tin, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R′ is H or a lower alkyl groupand wherein R_(ph) represents an unsubstituted or substituted phenylgroup with the phenyl substituents being chosen from any of thenon-phenyl substituents defined from R₁ and R₂ above, tetrazole andoxadiazole of the form:

wherein R′ is H or a lower alkyl group, or a triazene of the form:

viii) if the compound of Formula I is:

wherein R₅ is COOR′ (wherein R′ is H or lower alkyl), then at least oneof R₁-R₄, R₆ or R₇ is independently selected from the group consistingof F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R′ Hor a lower alkyl group, unless indicated, and wherein R_(ph) representsan unsubstituted or substituted phenyl group with phenyl substituentsbeing chosen from any of the non-penyl substituents defined from R₁ andR₂ above, tetrazole and oxadiazole of the form:

or a triazene of the form:

ix) if the compound of Formula I is:

wherein both R₅ are N(R′)₂ (wherein R′ is a lower alkyl group), then atleast one of R₁-R₄, R₆ or R₇ is independently selected from the groupconsisting of F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′, whereinn=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F,CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′,tri-alkyl tin, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), wherein R′ is Hor a lower alkyl group, unless indicated, and wherein R_(ph) representsan unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined from R₁ and R₂ above, tetrazole and oxadiazole of the form:

or a triazene of the form:

x) if the compound of Formula I is:

wherein at least one of R₃, R₅ or R₇ is a lower alkyl group, Cl, or OR′(wherein R′ is a lower alkyl group), then at least one other of R₂-R₇ isindependently selected from the other of R₁-R₇ is independently selectedfrom the group consisting of F, Cl, Br, I, (CH₂)_(n)OR′, wherein n=1, 2,or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN,C(═O)—R′, N(R′)₂, NO₂, O(CO)R′, OR′ (wherein R′ is H), SR′, a tri-alkyltin, R_(ph), CR′═CR′R_(ph), CR₂′—CR₂′R_(ph), wherein R′ is H or a loweralkyl group, unless indicated, and wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂ above, tetrazole and oxadiazole of the form:

wherein R′ is H or a lower alkyl group, or a triazene of the form:

xi) if the compound of Formula I is

wherein exactly two to R₂ are OR′ (wherein R′ is a lower alkyl group),then R₅ is selected from the group consisting of H, F, Cl, Br, I, or alower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F,O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, tri-alkyl tin, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph), wherein R′ is H or a lower alkyl group, unlessindicated, and wherein R_(ph) represents an unsubstituted or substitutedphenyl group with the phenyl substituents being chosen from any of thenon-phenyl substituents defined from R₁ and R₂ above, tetrazole andoxadiazole of the form:

or a triazene of the form:

xii) if the compound of Formula I is:

wherein R₅ is N(R′)₂ (wherein R′ is a lower alkyl group) and R₂, R₄, R₆,and R₇ are all H, then R₃ and R₇ are independently selected from thegroup consisting of F, Cl, Br, I, (CH₂)_(n)OR′, wherein n=1, 2, or 3,CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′ SR′, COOR′, tri-alkyl tin,R_(ph), R_(ph), CR′═CR′—R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph),wherein R′ is H or a lower alkyl group, unless indicated, and whereinR_(ph) represents an unsubstituted or substituted phenyl group with thephenyl substituents being chosen from any of the non-phenyl substituentsdefined from R₁ and R₂ above, tetrazole and oxadiazole of the form:

or a triazene of the form:

IB has the following structure:

wherein: each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently isdefined the same as R₁ above, provided that: xiii) if the compound ofFormula I is:

wherein exactly two to R₂ are a lower alkyl group, then at least one ofR₁, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅ or R₁₆ is independently selected fromthe group consisting of F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′,wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F,O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, tri-alkyl tin, R_(ph), CR′═CR′R_(ph), CR₂′—CR₂′—R_(ph),wherein R′ is H or a lower alkyl group and wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂ above, tetrazole and oxadiazole of the form:

or a triazene of the form:

xiv) if the compound of Formula I is:

wherein exactly two to R₂ are a lower alkyl group, then at least one ofR₁, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅ or R₁₆ is independently selected fromthe group consisting of F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′,wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F,O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, tri-alkyl tin, R_(ph), CR′═CR′R_(ph), CR₂′—CR₂′—R_(ph),wherein R′ is H or a lower alkyl group and wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂ above, tetrazole and oxadiazole of the form:

or a triazene of the form:

IC has the following structure:

wherein: each of R₁₇, R₁₈, R₁₉, R₂₀, or R₂₁ independently is defined thesame as R₁ above; ID has the following structure:

 wherein each of R₂₂, R₂₃, or R₂₄ independently is defined the same asR₁ above

 represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

wherein: each of R₂₅, R₂₆, or R₂₇ independently is defined the same asR₁ above

 represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

wherein: exactly one of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁, orR₃₂ independently is defined the same as R₁ above; IG has the followingstructure:

wherein: exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈ or R₃₉ independently is defined the same as R₁ above.
 2. Thecompound of claim 1, wherein at least one of the substituents R₁-R₇ andR₁₀-R₃₉ is selected from the group consisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br,¹⁸F, CH₂—CH₂—¹⁸F, O—CH₂—CH₂—¹⁸F, CH₂—CH₂—CH₂ ¹⁸F, O—CH₂—CH₂—CH₂—¹⁸F,¹⁹F, ¹²⁵I and a ¹¹C or ¹³C labeled moiety selected from the groupconsisting of a lower alkyl group, (CH₂)_(n)OR′, CF₃, CH₂—CH₂—F,O—CH₂—CH₂—F, CH₂—CH₂—CH₂—F, O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, (C═O)N(R′)₂,O(CO)R′, OR′, COOR′, R_(ph), CR′═CR′—R_(ph), and CR′2—CR′R_(ph).
 3. Thecompound of claim 1, wherein said compound binds to Aβ with adissociation constant (K_(D)) between 0.0001 and 10.0 μM when measuredby binding to synthetic Aβ peptide or Alzheimer's Disease brain tissue.4. A method for synthesizing a compound of claim 1, wherein at least oneof the substituents R₁-R₇ and R₁₀-R₃₉ is selected from the groupconsisting of ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, and ¹⁹F, comprising thestep of labeling a compound of claim 1, wherein at least one of thesubstituents R₁-R₇ and R₁₀-R₃₉ is a tri-alkyl tin or a triazene, byreaction of a compound of claim 1 with a ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br,¹⁸F, or ¹⁹F containing substance.
 5. A pharmaceutical composition for invivo imaging of amyloid deposits, comprising (a) a compound of claim 2,and (b) a pharmaceutically acceptable carrier.
 6. An in vivo method fordetecting amyloid deposits in a subject, comprising the steps of: (a)administering a detectable quantity of the pharmaceutical composition ofclaim 5, and (b) detecting the binding of said compound to amyloiddeposit in said subject.
 7. The method of claim 6, wherein said amyloiddeposit is located in the brain of a subject.
 8. The method of claim 6,wherein said subject is suspected of having a disease or syndromeselected from the group consisting of Alzheimer's Disease, familialAlzheimer's Disease, Down's Syndrome and homozygotes for theapolipoprotein E4 allele.
 9. The method of claim 6, wherein saiddetecting is selected from the group consisting of gamma imaging,magnetic resonance imaging and magnetic resonance spectroscopy.
 10. Themethod of claim 9, wherein said gamma imaging is either PET or SPECT.11. The method of claim 7, wherein said pharmaceutical composition isadministered by intravenous injection.
 12. The method of claim 7,wherein the ratio of (i) binding of said compound to a brain area otherthan the cerebellum to (ii) binding of said compound to the cerebellum,in said subject, is compared to said ratio in normal subjects.
 13. Apharmaceutical composition for the prevention of cell degeneration andtoxicity associated with fibril formation in amyloidosis-associatedconditions comprising a Chrysamine G derivative of claim 1 and apharmaceutically acceptable carrier.
 14. A method of detecting amyloiddeposits in biopsy or post-mortem human or animal tissue comprising thesteps of: (a) incubating formalin-fixed tissue with a solution of acompound of claim 1 to form a labelled deposit and then, (b) detectingthe labelled deposits.
 15. The method of claim 14 wherein said solutionis composed of 25-100% ethanol (the remainder being water) saturatedwith the compound.
 16. The method of claim 14 wherein said detecting iseffected by microscopic techniques selected from the group consisting ofbright-field, fluorescence, laser-confocal, and cross-polarizationmicroscopy.